Sen_G_ID Gene name Gene ID Category Cell name Tissue type Phenotype Aging type Experiment Description Target gene Regulatory type of target gene Target gene experiment Target gene description Regulatory pathway Regulatory type of pathway Pathway experiment Pathway description Species Experimental category Aging characteristic PMID Sen_G_0001 MYC 4609 protein coding TRE293 -- Aging Prevent Western blot//SA-β-gal activity assay TRE293 cells treated with c-Myc shRNA demonstrated significant changes in senescence and induced the p53/p21CIP1 pathway. BAG2 Downregulation Western blot//BrdU assay//SA-β-gal activity assay "In cells that demonstrated high levels of BAG2 in response to SP1 over-expression, transfection of c-Myc was shown to abrogate the BAG2 mRNA levels in a concentrationdependent manner." p53-p21 Upregulation Western blot TRE293 cells treated with c-Myc shRNA demonstrated significant changes in senescence and induced the p53/p21CIP1 pathway. Human HL cellular senescence 22146591 Sen_G_0002 NLRX1 79671 protein coding HCCLM3 -- Hepatocellular carcinoma Accelerate CCK-8 assay//SA-β-gal activity assay "We found that NLRX1 overexpression could hinder cell proliferation in HCCLM3 cells, while knocking down NLRX1 in Huh7 resulted in significant higher proliferation rates. NLRX1-OE significantly increased SA-β-gal activity, indicating a higher proportion of aging in NLRX1-OE cells compared to cells transfected with vector." p21 Upregulation Western blot//RT-PCR "We found that NLRX1-OE could effectively upregulate P21 expression, and expressions of downstream targets of P21 including CDK1 and CDK2 were significantly repressed due to high level of P21 expression caused by NLRX1 overexpression, while knocking down NLRX1 in Huh7 resulted in increased expression of CDK1 and CDK2." PI3K-Akt Downregulation Western blot "NLRX1-OE markedly inhibited the phosphorylation of mTOR, indicating that NLRX1 suppresses PI3K-AKT signaling." Human HL cellular senescence 29482578 Sen_G_0003 PRKAA1 5562 protein coding NIH-3T3 -- Aging Prevent SA-β-gal activity assay "Next, we observed that when the H2O2-treated cells were incubated with medium containing Met and BBR, there was a dose-dependent decrease in the percentage of SA-β-Gal-positive cells, which was similar to that caused by rapamycin treatment.As expected, when combined with CC, the effects of Met or BBR on senescence prevention were largely blunted when CC was coexisted, as indicated by the remarkable increase in SA-β-Gal-positive cells." LC3//p62 Downregulation//Downregulation Western blot "With Western blot assay, we found that both Met and BBR alleviated the H2O2-induced accumulation of the LC3 and p62 proteins, and this alleviation was consistent with the effect of rapamycin. " mTOR Downregulation Western blot "Moreover, we found that BBR treatment significantly suppressed mTOR phosphorylation in senescent cells, similar and even stronger than that induced by rapamycin and insulin as an mTOR activation control." Human L delay aging 26890602 Sen_G_0004 IGFBP7 3490 protein coding MDA-MB-231 -- Breast cancer Accelerate SA-β-gal activity assay//Flow cytometry//Cell morphological analysis "IGFBP7 treatment of MDA-MB-231 cells increased the percentage of cells in G1 (73.86%) compared to untreated cells (54.14%), with a consequent decrease in the percentage of cells in S phase (15.85% in IGFBP7-treated cells versus 33.8% in untreated controls).We found that MDA-MB-231 cells treated with IGFBP7 for 2 days underwent morphological changes characterized by cell aggregation with strong expression of senescence-associated β-galactosidase ." p21//p53 Upregulation//Upregulation Western blot "In agreement with this, we found that IGFBP7-treated MDAMB-231 cells had higher p21 and p53 expression than untreated cells." p38 MAPK Upregulation Western blot Levels of phosphorylated p38 MAPK were dramatically increased by IGFBP7 treatment. Human L apoptosis 21997538 Sen_G_0005 MEF2A 4205 protein coding HUVEC Human umbilical vein Cardiovascular disease Prevent Cell viability assay//SA-β-gal activity assay The percentage of senescent phenotype cells in MEF2A overexpression group was significantly lower than that in the empty plasmid group and the MEF2A mutant group (pc-gab-MEF2A-MV30).MEF2A overexpression in HUVECs significantly increased cell viability. p53 Downregulation Western blot The level of p53 was significantly decreased?in the MEF2A-overexpression HUVECs. PI3K-p-AKT-SIRT1 Upregulation qRT-PCR//Western blot "PIK3CA, PIK3CG, and SIRT1 mRNA levels were significantly up-regulated in the MEF2Aoverexpression HUVECs, and the protein levels of PIK3CA, PIK3CG, SIRT1, and p-Akt notably increased ." Human L Others 31182679 Sen_G_0006 MIR584 693169 ncRNA "MGC803,SGC-7901" -- Gastric cancer Accelerate SA-β-gal activity assay//Western blot "The miR-584- 5p-mimic-transfected cells showed strong blue SA-β-gal staining, while only scattered SA-β-gal-positive cells were detected in miR-584-5p-inhibitor-transfected group compared with the corresponding control cells.The expression of p53, p21 and p16 was enhanced after miR584-5p-mimic-transfection. Conversely, the expression of p53, p21 and p16 was reduced after miR-584-5p-inhibitor transfection." WWP1//p53 Downregulation//Upregulation SA-β-gal activity assay//Western blot//Flow cytometry "Intriguingly, co-transfection of MGC803 cells with miR-584-mimic and either LV-WWP1 or LV-NC showed that ectopic expression of WWP1 effectively reversed the promotion of cellular senescence resulting from miR-584-5p overexpression. Similarly, the effects of miR584-5p knockdown in SGC7901 were counteracted by WWP1 downregulation. The induction of SA-β-Gal positively stained cells by miR-584-5p was also reduced by treatment of pifithrin-α, indicating the involvement of p53 in miR-584-5p-induced cellular senescence.In accordance with the analysis of GC tissues, Western blot analysis showed an inverse correlation between WWP1 expression and miR-584-5p expression in GC cell lines and GES-1 cells.The expression of p53, p21 and p16 was enhanced after miR-584-5p-mimic-transfection." TGFβ Activation Western blot//Immunofluorescence "Western blot analysis of the expression of WWP1 and key proteins involved in TGFβ signaling pathway showed that the protein levels of TβR1, Smad2, Smad4, and the CDK inhibitor p15 were increased after miR-584-5p-mimic transfection, while the opposite effects were observed after miR-584-5p-inhibitor transfection.However, the expression of TβR1, Smad2, Smad4, and p15 was decreased in LY364947 treated cells compared with that of the control group. Furthermore, immunofluorescence assays revealed a dramatically positive correlation between the levels of miR-584-5p and TGFβ." Human HL cellular senescence 28431583 Sen_G_0007 TP53 7157 protein coding EJ -- Aging Accelerate Knockdown//SA-β-gal activity assay "The adenovirus-mediated expression of p53 in EJ p53-null human bladder cancer cells promoted remarkable morphological changes and senescence-associated β-galactosidase (SA-β-gal) activity, which are features of premature senescence phenotypes,within 6 days. Irreversible growth arrest, which is characteristic of cellular senescence, was also confirmed by a marked reduction in cell growth and decreased number of cells in the S-phase upon p53 expression." p21//Akt --//Activation SA-β-gal activity assay//Western blot "P53-mediated Akt activation and the inhibition of p53-induced SA-β-gal activity by LY294002 treatment were also observed in H1299 human lung cancer cells .Akt and p21 separately regulate p53-induced ROS increases and cell cycle arrest, respectively.suppression of the induction of p21 expression successfully inhibited the p53-induced increase in SA-β-gal activity and morphological changes. In addition, the p53-induced loss of proliferation and cell cycle arrest were inhibited by the suppression of p21 induction .We used a p21 shRNA retrovirus to specifically suppress the p53-mediated induction of p21 expression and cormnfied that its expression was effectively suppressed using Western blot analyses." NF-κB-NOX4 -- SA-β-gal activity assay//ChIP//RT-PCR "NOX4 knockdown resulted in the significant suppression of p53-induced SA-β-gal activity but did not affect the loss of proliferation.Importantly, the chromatin immunoprecipitation (ChIP) assay results indicated that NF-kB binding to the putative binding site on the NOX4 promoter was enhanced by p53 expression, but this NF-kB binding was inhibited by BAY 11-7082 treatment. Furthermore, Akt inhibition by either LY294002 or Akt inhibitor IV treatment si gnificantly inhibited the increase in NF-kB binding in response to p53 expression, indicating that Akt activity is required for NF-kB binding to the NOX4 promoter." Human L cellular senescence 28691365 Sen_G_0008 NAMPT 10135 protein coding MEF -- Aging Prevent SA-β-gal activity assay "Over- expression of NAMPT in MEF cells slows down cellular senescence.Percentage of cells that is positive for SA- β- Gal activity in all Wt- MEF cells was dramatically increased from passage 6, consistent with the timing of CCP in Wt- MEF cells.we measured the expression level of several senescence marker genes. p16INK4a, p19ARF, and p21CIP1 are cell cycle inhibitors and their expression levels increase when cells enter senescence." SIRT1 Upregulation qRT-PCR "We finally examined whether SIRT1 activity is enhanced in response to NAMPT over- expression. Although SIRT1 has been shown to regulate cellular proliferation and senescence, this function is dependent on the robustness of the intrinsic NAMPT activity and NAD+ content. SIRT1 is also vital in regulating FOXO- mediated activation of antioxidant enzymes including SOD2 and Catalase. As we shown above, NAD+ content in Tg- MEF was higher than that in Wt- MEF cells. SIRT1 activity in Tg- MEF was approximately threefold higher than that in Wt- MEF cells, in line with the observed SOD2 and catalase up regulation." -- -- -- -- Human L cellular senescence 29178516 Sen_G_0009 SIRT1 23411 protein coding WI-38 -- Aging Prevent SA-β-gal activity assay//BrdU assay "As observed for the growth curves,expression of SIRT1 alone does not significantly affect the percentage of cells in S phase or the percentage of SA-β-gal-positive cells when compared with control infected cells. However, expression of PML IV results in a striking decrease of BrdU-positive cells and a huge increase in SA-β-gal Activate cells, as reported previously. Double-infected PML IV–SIRT1 cells exhibit a significant increase in the percentage of cells in S phase as compared with PML-infected cells, in addition to a marked decrease in the percentage of SA-β-gal-positive cells. " p53 Binding Pull-down assay "We performed pull-down assays with either full-length GST–p53 or a series of GST–p53 fusions expressing only the N-terminal, middle or C-terminal regions of the protein, and in vitro translated SIRT1. We observed specific binding of SIRT1 to full-length p53 as well as to GST–p53(290–393), while weaker binding was seen with GST–p53(90–290) and no binding occurred with the N-terminal p53 fusion." -- -- -- -- Human HL cellular senescence 12006491 Sen_G_0010 CDK5 1020 protein coding Saos-2 -- Aging Accelerate SA-β-gal activity assay//Knockdown "SiCDK5 or dnCDK5 partially inhibited this activity, and interestingly, CDK5 overexpression enhanced the number of SA-β-Gal-positive cells." Rac1 Downregulation SA-β-gal activity assay "In the presence of activated Rac1, most pRb induction of SA-β-Gal was compromised, a phenotype that was exacerbated by expression of dnCDK5, roscovitine, or siCDK5 treatment . Coexpression of CDK5 with pRb and RacV12 resulted in a partial rescue of SA-β-Gal expression, suggesting that CDK5 might be antagonizing some aspect of Rac1 function. However, in cells transfected with RB and RacN17, coexpression of dnCDK5 and siCDK5 or roscovitine treatment in no way inhibited pRbinduced SA-β-Gal expression." -- -- -- -- Human L cellular senescence 15024070 Sen_G_0011 MORC3 23515 protein coding WI-38 -- Aging Accelerate SA-β-gal activity assay//Western blot "The percentage of SA-β-Gal–positive cells infected with the MORC3-retrovirus vector (51.0±5.1%) was significantly greater than that of controls infected with vector alone (2.3±0.8%). MORC3 expression also strongly induced p16, another senescence marker." p53 Activation Luciferase reporter assay "FLAG-MORC3 expression enhanced transcriptional activation by endogenous p53 of a reporter containing 12 p53-responsive elements (PG12S) in a dose-dependent manner, but not activation of a reporter containing 14 mutated responsive elements (MG14S)." -- -- -- -- Human L cellular senescence 17332504 Sen_G_0012 IGFBPL1 347252 protein coding MCF-7 -- Aging Accelerate Cell counting//SA-β-gal activity assay//Flow cytometry "Cell proliferation of MCF-7 cells was severely inhibited following IGFBP-rP1 transfection,whereas there were indistinguishable differences in cell proliferation between the control vector-transfected and the untransfected cells. On day 7, the IGFBP-rP1-transfected MCF-7 cells had an enlarged flat morphology,characteristic of senescent cells. At the same time, a significant increase in senescence-associated-β-galactosidase (SA-β-gal) activity was detected in IGFBP-rP1-transfected MCF-7 cells, as compared with control cells transfected with empty vectors or the cells that had not been transfected at all. Addition of CM from IGFBP-rP1-transfected cells to untransfected MCF-7 cells blocked cell proliferation and increased SA-β-gal activity, whereas CM from the control vector (pEGFP-N1)-transfected or untransfected cells failed to inhibit cellular proliferation and induce senescence.A significant increase in the cell population at the G /G1phase of the cell cycle was detected in IGFBP-rP1-transfected MCF-7 cells, which is one of the typical phenotypes in cellular senescence." p21 Upregulation Knockdown//Western blot//Cell counting//SA-β-gal activity assay "Basal and IGFBP-rP1-inducible p21 levels deceased dramatically in p21 knockdown MCF-7 cells.Accordingly, IGFBP-rP1-mediated pRb dephosphorylation was substantially reduced in these cells. We also examined cell proliferation and cellular senescence in response to IGFBP-rP1 in parental and p21 knockdown MCF-7 cells. The results showed that cell proliferation block and increased SA-β-gal activity in response to IGFBP-rP1 were partially reversed by p21 knockdown. These results suggest that cellular senescence induced by IGFBP-rP1 is mediated at least in part by p21 up-regulation." -- -- -- -- Human L cellular senescence 22392074 Sen_G_0013 GADD45G 10912 protein coding "SK-Hep-1,SMMC-7721" -- Hepatocellular carcinoma Accelerate SA-β-gal activity assay//Cell morphological analysis "In agreement with previous observation, we confirmed that the induction of GADD45G expression in Sk-Hep1 cells (Tet-on-GADD45G-Sk-Hep1) and SMMC-7721 cells (Tet-on-GADD45G-SMMC-7721) resulted in characteristic morphological changes common in senescent cells and a dramatic increase in the proportion of SA-β-Gal-staining positive cells." SIP1 Upregulation qRT-PCR//Western blot "Surprisingly, we found that the levels of SIP1 mRNA were significantly elevated in the cells upon GADD45G induction.The change in SIP1 mRNA expression was also reflected at the protein levels in Sk-Hep1 and SMMC-7721 cells upon GADD45G induction. In addition, GADD45G-induction of SIP1 expression similarly occurred in H-Ras V12-transformed mouse p53-/- liver progenitor cells (LPC-H-Ras V12 cells)." -- -- -- -- Human HL cellular senescence 26378039 Sen_G_0014 HMGA2 8091 protein coding WI-38 -- Retinoblastoma Accelerate SA-β-gal activity assay//qPCR//Western blot "We found that the ectopic overexpression of HMGA2 led to elevated SA-β-galactosidase staining and acquisition of the SAHF-like chromatin foci in the nuclei. Meanwhile, the expression levels of the senescence-related proteins, such as p16, p21, p53 and phosphorylated p53, were prominently increased. The transcription level of HDM2 that reduces the stability of p53 protein was decreased together with a slightly up-regulated p14ARF, an inhibitor of HDM2, in WI38 cells expressing HMGA2 . Apparently, these results indicate that HMGA2 can induce the senescence phenotypes in WI38 cells." E2F Downregulation Western blot "Surprisingly, both HMGA2 and Rb were detected in the same protein complex in our model (data not shown), together with the down-regulation of E2F target genes in WI38 cells in response to HMGA2 overexpression." Rb -- Western blot//qPCR//RT-PCR//Immunofluorescence "To further test whether Rb is a crucial factor in SAHF formation induced by HMGA2, E7 protein was used as the specific inhibitor of Rb , and the effect of E7 protein on the degradation of Rb was confirmed by Western blotting . Interestingly, we found that E7 protein was able to partly abolish the SAHF formation induced by HMGA2, as revealed by cellular immunofluorescence and decreased percentage of cells with SAHF .Furthermore, as another inhibitor targeted to Rb, SV40 had a similar effect to decrease the HMGA2-induced SAHF formation ." Human L cellular senescence 23969248 Sen_G_0015 RRAD 6236 protein coding NHF -- Aging Prevent Knockdown//SA-β-Gal activity assay We found that RRAD knockdown increased the ratio of senescent cells in both H2O2- and IR-induced senescence as determined by SA-β-gal staining assay and BrdU incorporation assay. p53//NF-κB --//-- Knockdown//CHIP-Seq//qRT-PCR "We established p53 knockdown NHFs by a lentiviral based approach for the following functional assay. In OIS, p53 knockdown diminished H-Ras-induced RRAD up-regulation.p53 knockdown also abolished H 2O2-induced RRAD expression.In a published ChIP-Seq data set of IMR-90 cells, we noted that both p53 and NF-κB bind to the RRAD promoter region in adjacent sites." -- -- -- -- Human HL cellular senescence 30391675 Sen_G_0016 ING1 3621 protein coding 2BS -- Aging Accelerate SA-β-gal activity assay//SAHF//Immunofluorescence "The results showed that 2BS cells overexpressing of p33ING1befficiently induced the expression of SA-β-gal activity, flattened senescent cell morphology, and SAHF DNA staining pattern which is indicated by co-localization with H3K9Me3." p16 Upregulation qPCR//Western blot//Luciferase reporter assay "qPCR and Western blot results showed that introduction of p33ING1b upregulated p16INK4amRNA and protein levels.Moreover, p16INK4apromoter activity was upregulated by p33ING1b.The results showed that cells treated with p33ING1balone revealed strong SA-β-gal staining and p16 increasing, whereas cells doubly treated with p33ING1b and p16-shRNA reversed the senescence phenotypes. As expected, p16-shRNA-infected cells displayed young phenotypes." -- -- -- -- Human L cellular senescence 21896275 Sen_G_0017 TWIST 100303576 protein coding SW48 -- Aging Prevent SA-β-gal activity assay//BrdU assay Higher percentage of SA-β-gal-positive cells was found in the Sh-TWIST transfectants (filled columns) compared with the controls (open columns) in response to both agents in a dose-dependent manner. This was associated with a decreased cell proliferation rate examined by BrdUrd incorporating assay.These results suggest that suppression of TWIST expression promotes cellular senescence in PrEC. p14//cH2AX Downregulation//Upregulation Western blot "We also found that the p14ARF expression levels were much lower in the TWIST over-expressing cells, either before or after exposure to H2O2 and CP (right panels), compared with the vector control, confirming the negative effect of TWIST on p14ARF. In addition, the p53 and p21 levels were lower in the TWIST over-expressing cells compared with the vector control, especially after drug treatment. Furthermore, we found that the level of cH2AX was higher in the TWIST over-expressing cells either before or after drug treatment compared with the vector control, which was associated with lower levels of phosphorylated Chk1/2 proteins, especially after drug treatment." Mdm2-p53 -- Western blot "Western blotting analysis showed that the expression levels of p14ARF were much higher in the Sh-TWIST transfectants after exposure to H2O2 or CP in a dose-dependent manner. In contrast, the level of MDM2 was decreased, which was associated with up-regulation of p53 and p21, suggesting that inactivation of TWIST promotes p14ARFMDM2-p53-mediated DNA damage response pathway." Human L cellular senescence 17690110 Sen_G_0018 CAV1 857 protein coding MEF -- Aging Accelerate SA-β-gal activity assay//Western blot "In contrast, wild type MEFs over-expressing caveolin-1 developed a senescent phenotype 4 days after oxidative stress, as determined by senescent-associated β-galactosidase staining and p21 upregulation." SIRT1 Binding Pull-down assay "To determine whether Sirt1 binds directly to caveolin-1, we performed GST pulldown assays using recombinant Sirt1. We found that human recombinant Sirt1 bound to caveolin 1 (residues 82–101)-GST.The incubation with the purified GST-Cav-1 (82–101) peptide inhibited Sirt1 activity by- 45% compared to GST alone." p53 -- Pull-down assay//IP//Western blot "We found that human recombinant Sirt1 bound to caveolin 1. However, Sirt1 strongly interacted with caveolin-1 72 hours after stimulation of WI-38 cells with sublethal doses of hydrogen peroxide that we have previously shown induce premature senescence.In contrast, free radicalinduced acetylation of p53 was significantly inhibited in cells lacking caveolin-1. Interestingly, downregulation of Sirt1 by siRNA enhanced oxidant-induced acetylation of p53 in caveolin-1 null MEFs to levels seen in wild type MEFs." Human L cellular senescence 25512378 Sen_G_0019 NQO1 1728 protein coding 2BS -- Aging Accelerate SA-β-gal activity assay//qRT-PCR//Cell proliferation assay//BrdU assay "NQO1 depletion resulted in continuous cell growth compared with corresponding control lentiviral vector (Cont) infected cells . In addition, the morphology changed during the OIS, with senescent cells exhibiting the typical enlarged and flattened shape. Five days after introducing RasG12V, NQO1 depleted 2BS cells showed a different morphology compared with control 2BS cells. Accordingly, we found that 2BS cells with NQO1 depletion reduced with SA-β-gal staining after expressing RasG12V. We observed that knockdown of NQO1 resulted in an increased percentage of 2BS cells with BrdU incorporation after expressing RasG12V. This result suggests that NQO1 depletion delayed the onset of RasG12V-induced cellular senescence." p53 Binding Western blot "To test the relevance of NQO1-p53 binding in OIS, p53 was immunoprecipitated in 2BS cells. Western blot analysis verified that NQO1 specifically interacted with p53 in premature senescence." Nrf2-Keap1 -- Western blot//CHIP//qPCR//Knockdown//Luciferase reporter assay "MAF and KEAP1 were immunoprecipitated from 2BS cells, and western blot analysis verified that during OIS NRF2 was dislocated from KEAP1 and bound with MAF, suggesting that OIS activates NRF2 and results in NRF2-mediated up-regulation of NQO1. Next, we detected whether NRF2 bound to the endogenous NQO1 promoter by chromatin immunoprecipitation (ChIP) assay. Interaction between NRF2 and the NQO1 promoter region was detected by qPCR. As expected, interaction between NRF2 and NQO1 promoter only observed in senescent 2BS. We found that knockdown of NRF2 diminished its interaction with the NQO1 promoter. In addition, we also determined whether NRF2 affected NQO1 promoter activity with ARE by the luciferase reporter assay." Human HL cellular senescence 26078718 Sen_G_0020 RSL1D1 26156 protein coding 2BS -- Aging Prevent SA-β-gal activity assay "No SA-β-gal activity was observed in CSIG transfected 2BS and early-passage cells, whereas CSIG-CT transfected 2BS cells were strongly stained, as were late-passage (senescent) 2BS control cells." PTEN Downregulation Western blot "As expected, CSIG overexpression diminished PTEN abundance." -- -- -- -- Human L cellular senescence 26686419 Sen_G_0021 S100A13 6284 protein coding IMR-90 -- Aging Accelerate Knockdown//SA-β-gal activity assay "S100A13 overexpression promotes, whereas S100A13 knockdown delays replicative cellular senescence in IMR9 cells. S100A13 mutant significantly decreased the percentage of SA-β-gal positive staining cells." IL-1 Upregulation Flow cytometry//Immunoblotting FACS analysis of cell surface-bound IL-1α showed that S100A13 overexpression significantly enhanced IL-1α levels compared to empty vector control cells. -- -- -- -- Human L cellular senescence 30670674 Sen_G_0022 TRIB2 28951 protein coding LoVo -- Colorectal cancer Prevent Knockdown//Flow cytometry//SA-β-gal activity assay//Cell morphological analysis "TRIB2 knockdown cells showed higher rates of flat and enlarged senescence-like morphology.Cell cycle distribution of the cells transfected with TRIB2-specific siRNA were determined by flow cytometry.TRIB2 knockdown dramatically increased the G0/G1-phase ratios and reduced the S-phase ratios. The level of SA-β-gal (a biomarker of senescence) was measured, and data showed that the percentage and strength of SA-β-gal-positive cells were significantly increased in TRIB2 knockdown condition." -- -- -- -- AP4-p21 -- Western blot//RT-PCR//Luciferase reporter assay//IP//CHIP "Silencing TRIB2 dramatically up-regulated p21 expression, inhibited cell growth, induced cell cycle arrest and enhanced cellular senescence. The luciferase assay indicated TRIB2 inhibited p21 promoter activities in an AP4-dependent manner. We then performed CCK8 and SA-β-gal staining analysis to examine the effects of AP4 on TRIB2-mediated functions, and found that silencing AP4 could significantly block TRIB2 overexpression-promoted SW48 and LoVo cells growth and enhance TRIB2 overexpression-inhibited cellular senescence. We carried out co-IP assay using SW48 cells or HEK 293T cells co-transfected with Flag-AP4 and His-TRIB2. AP4 and TRIB2 were found to precipitate with each other.The results showed that overexpression of AP4 suppressed and co-transfection of TRIB2 further suppressed the activities of p21 promoter , which indicated TRIB2 enhanced the function of AP4." Human L cellular senescence 30541550 Sen_G_0023 LBR 3930 protein coding TIG-7 -- Aging Prevent Knockdown//SA-β-Gal activity assay "We also found that the cells transfected with the LBR knockdown vector were stained with SA-β-gal, which observation suggested that knockdown of LBR induced cellular senescence in TIG-7 cells." -- -- -- -- -- -- -- -- Human L cellular senescence 30615890 Sen_G_0024 PTEN 5728 protein coding MCF-7 -- Aging Prevent Knockdown//SA-β-Gal activity assay The SA-β-Gal positivity of PTEN KO MEFs was dramatically attenuated in Torin1-treated cells compared to Rapa-treated cells. -- -- -- -- p53-p21 -- Western blot PTEN-depleted MCF7 cells displayed prematurely senescent phenotypes in a p53/p21-dependent manner. Human L cellular senescence 30337688 Sen_G_0025 MDM2 4193 protein coding HCT116 -- Werner syndrome Accelerate SA-β-Gal activity assay "Ectopic expression of WRN attenuates the senescent phenotype induced by Etoposide,as shown by senescence-associated β-galactosidase (SA-β-Gal) activity." WRN -- IP WRN and MDM2 association was detected in co-transfected 293T cells by reciprocal immunoprecipitations. -- -- -- -- Human L cellular senescence 30532073 Sen_G_0026 ASPH 444 protein coding "FOCUS,HuH-7,HepG2" -- Hepatocellular carcinoma Prevent Knockdown//SA-β-Gal activity assay "Knockdown or knockout of ASPH, caused substantial increase in β-gal staining as a marker for the cells undergoing senescence." GSK3β -- Western blot//IP//Immunoblotting We found a significant increase of phospho-GSK3β(inactivated form) with equal protein expression of GSK3β when comparing the shASPH to the shLuc control.Direct protein-protein interaction between GSK3βand ASPH was demonstrated by co-immunoprecipitation (IP).Immunoblotting with anti-GSK3β revealed that GSK3β bound to ASPH following co-transfection with WT-GSK3βand ASPH. -- -- -- -- Human L cellular senescence 26683595 Sen_G_0027 RXRA 6256 protein coding MRC-5 -- Aging Prevent Knockdown//SA-β-Gal activity assay "RXRA knockdown decreased cell proliferation as we observed a drop in the expression of the cell proliferation marker Ki‐67 and a decrease in the number of cells .Importantly, RXRA knockdown also caused an increased SA‐β‐galactosidase activity." p53 -- Knockdown//Western blot "Using the same approach,we further showed that cellular senescence induced by RXRA knockdown was also dependent on MCU and p53." ITPR2-MCU -- Knockdown//qRT-PCR//Western Blot//SA-β-Gal activity assay "To assess whether this senescent phenotype was dependent on ITPR2, we knocked down ITPR2 together with RXRA.ITPR2 knockdown impaired the upregulation of CDKN1A and GDF15 . The proliferation arrest and the increase in SA‐β‐galactosi-dase activity triggered by RXRA knockdown were alsoimpaired. Similar observations were made using either a siITPR2 pool or individual siRNAs targeting ITPR2. Using the same approach, we further showed that cellular senescence induced by RXRA knockdown was also dependent on MCU and p53." Human HL cellular senescence 30216632 Sen_G_0028 LRRK2 120892 protein coding SH-SY5Y -- Parkinson’s disease Accelerate Western blot Up-regulated β-galactosidase protein levels in G2019S-mediated phosphomimetic p53 and overexpression of p21 or G2019S LRRK2. -- -- -- -- p53-p21 Upregulation Western Blot//SA-β-Gal activity assay "We observed a significant increase in β- gal protein level with the expression of T304/377D p53 (TD-p53) compared to that in the vector and WT-p53 in dSH-SY5Y cells . In addition, the overexpression of p21 and G2019S LRRK2 in dSH-SY5Y cells resulted in significant increases in β-gal protein expression. We found that ectopic expressions of eitherred fluorescence protein (RFP)-TD-p53 or RFP-p21 elevated GlycoGreen fluorescence by 2–3 folds." Human L cellular senescence 30712480 Sen_G_0029 TSHB 7252 protein coding -- Adipose Aging Prevent RT-PCR//Telomere length assay "In euthyroid morbidly obese subjects,both SAT and VAT TSHB gene expression was negatively associated with markers of AT senescence (TNF , TP53, BAX and DBC1). Confirming these findings, SAT TSHB mRNA was positively linked to telomere length in a subgroup of consecutive subjects , suggesting decreased senescence with less exposure to thyroid hormones. Furthermore, SAT TSHB mRNA was negatively correlated to specific senescence-associated glucosylceramides." -- -- -- -- -- -- -- -- Human L cellular senescence 30448824 Sen_G_0030 FXR1 8087 protein coding "UMSCC74A,UMSCC74B" -- Oral cancer Prevent Knockdown//Flow cytometry//SA-β-Gal activity assay "To investigate the underlying mechanism of reduction in number of viable cells following FXR1 KD, we performed cell cycle analysis of the FXR1 KD and control cells by flow cytometry. FXR1 KD induces cell cycle arrest in G /G1 providing evidence that FXR1 might regulate cell division in oral cancer.Silencing of FXR1 in multiple oral cancer cells also promotes senescence by positive SA-β-gal staining." TERC -- Luciferase reporter assay "When full-length and truncated TERC (28 base deletion at 5’-end,TERCmut） are used for luciferase assay,TERC exhibits increased luciferase activity in control cells compared to TERCmut. However, in FXR1 KD cells, TERC exhibits a decreased luciferase activity compared to TERCmut indicating that FXR1 binds to specific G-rich sequences in these RNAs and possibly controls their turnover.Silencing of FXR1 reduces the level of TERC and subsequently interferes with the telomerase activity." p53-p21 -- RT-PCR//Western Blot Upregulation ofp53 and p21 protein levels are observed in the absence of FXR1 in WT p53 cell line.RT-PCR、Western Blot：overexpressed FXR1 in HNSCC destabilizes p21 mRNA and reduces its protein expression.concurrent overexpression of p21 and silencing of TERC significantly promoted senescence evidenced by staining of SA-β-gal. Human HL cellular senescence 27606879 Sen_G_0031 CDKN2A 1029 protein coding DFC -- Aging Accelerate SA-β-Gal activity assay//Western blot P16 gene silencing reduces the number of senescent cells.Only the protein expression of P16 in DFCs increased successively and significantly while the expression of all other examined cell cycle proteins including that of P21 was down-regulated significantly at higher cell passages. -- -- -- -- -- -- -- -- Human HL cellular senescence 28770470 Sen_G_0032 SIRT6 51548 protein coding 2BS -- Aging Prevent Western blot//RT-PCR//Colony formation?assay//Flow cytometry "Western blot analysis revealed that the expression of SIRT6 was high in young cells, but decreased significantly during cellular senescence. Consistently, RT-PCR analysis revealed that mRNA levels of SIRT6 decreased in senescent cells .Another hallmarker of senescent cells is the ability of colony formation. We observed that 2BS cells overexpressing SIRT6 formed more colonies than control cells. However, SIRT6 shRNA-infected cells showed less colonies compared to control cells .It was obvious that SIRT6 shRNA-infected 2BS cells entered G1 cell cycle arrest." p27 -- Western blot//RT-PCR//SA-β-gal activity assay//Co-IP//Pull-down assay "In addition,SIRT6 silencing did not alter the protein levels of p16, p21, p53 and PTEN, but markedly increased p27 protein levels. Moreover, the real-time PCR analysis demonstrated similar p27 mRNA levels in both SIRT6-overexpressing and knockdown cells, suggesting that SIRT6 might decrease the p27 protein levels but not the mRNA levels.2BS cells co-expressing pLPC,pBabe-p27 exhibited G1 arrest and higher SA-β-gal ctivity. Concurrently, the senescent markers of SIRT6-p27 appeared to be comparable with that of pLPC-pBabe control cells.Thus,the decrease of p27 mediated by SIRT6 is required for the restriction of cellular senescence. Colocalization experiments showed that both SIRT6 and p27 mainly colocalized in the nucleus. To further confirm the interaction of p27 and SIRT6, we performed a GST pull down assay using in vitro transcribed and translated p27 or SIRT6 and purified GST-SIRT6 or GST-p27.SIRT6 displayed a strong association with p27 in vitro." -- -- -- -- Human HL cellular senescence 27794562 Sen_G_0033 TIMELESS 8914 protein coding 2BS -- Aging Prevent Western blot//qRT-PCR "The expression of circadian protein TIMELESS was down-regulated during the onset of OIS, while other members of circa-dian clock showed no significant changes in their expression.The downregulation of TIMELESS was also confirmed inreplicative senescence and H2O2 induced senescence,suggesting TIMELESS downregulation might be a commonevent happened during cellular senescence." E2F1 -- Knockdown//Luciferase reporter assay//CHIP//qRT-PCR//Western blot "Chromatin immunoprecipitation (ChIP) assay was utilized to confirm the binding of E2F1 to the endogenous TIMELESS promoter.Interaction between E2F1 and TIMELESS promoter region was detected by qRT-PCR.As expected, interaction between E2F1 and TIMELESS promoter was observed in young 2BS cells.Furthermore, the mutation in E2F1 binding site of TIMELESS promoter region significantly reduced the luciferase activity in young 2BS cells.Knockdown of E2F1 by a sh-E2F1 lentiviral vector significantly down-regulated TIMELESS mRNA level in young 2BS cells.All above data support that E2F1 could bind to TIMELESS promoter and regulated the expression in young 2BS cells." -- -- -- -- Human L cellular senescence 30100061 Sen_G_0034 TSLP 85480 protein coding BEAS-2B -- Asthma Accelerate SA-β-gal activity assay//Western blot "Specifically,senescent cells increased >15 fold upon treatment with 1.5ng/ml TSLP.There was increased expression of SA-β-gal and decreased Ki67 expression in BEAS-2B cells upon TSLP treatment." -- -- -- -- -- -- -- -- Human L cellular senescence 24167583 Sen_G_0035 UHRF1 29128 protein coding "2BS,IMR-90" -- Aging Prevent SA-β-gal activity assay//CCK-8 assay//EdU assay//Knockdown Cell proliferation rates decreased in UHRF1 shRNA-infected cells. Growth curves of UHRF1 shRNA-infected 2BS cells or IMR9 cells were determined by CCK8 assay.The percentage of cells incorporating EdU also decreased in UHRF1 shRNA-infected 2BS cells or IMR9 cells. SA-β-gal activity staining increased in UHRF1 shRNA-infected cells. Representative microscopic view of 2BS cells or IMR9 cells infected with the indicated vectors with SA-β-gal activity staining. PIM1 -- IP "In reciprocal immunoprecipitations, PIM1 was observed presentingin UHRF1 immunoprecipitates." -- -- -- -- Human HL cellular senescence 28394343 Sen_G_0036 WNT7A 7476 protein coding A549 Lung Lung cancer Accelerate SA-β-gal activity assay//CCK-8 assay "Histological analysis of the lung sections of the wild-type and Wnt7a-null mice showed no difference in basal staining for SA-β-gal activity. However, a large number of SA-β-gal-positive cells, predominantly in the tumor area, were observed in lung sections of the wild-type mice after urethane treatment, whereas Wnt7a-null mice,on the contrary, showed reduced or no SA-β-gal staining." -- -- -- -- SKP2-p27-pRb -- Western Blot "Consistent with the changesin SKP2 and p27kip1 expression, the levels of phosphorylated(p-Rb-S780) retinoblastoma proteins in Wnt7a-null mice were dramatically increased as well . Cyclin D1, which is critical for Ser780 phosphorylation of Rb ,was also upregulated in Wnt7a-null mice, along with CDK2 andCDK6, components of the CDK/cyclin complex . Similarresults were obtained when using the lung lysates of wild-typeand Wnt7a-null in FVB/NJ mice." Human HL cellular senescence 25728679 Sen_G_0037 SPHK1 8877 protein coding Adipose stromal cell -- Aging Prevent SA-β-gal activity assay//BrdU assay "A SPHK1 inhibitor, significantly reduced the incorporation of BrdU into cellular DNA compared to control cells,indicative of the lowered proliferative ability.As with high-passage cells,SPHK1-depleted or SKI-treated hAD-SCs underwent accelerated cellular senescence as evidenced by higher SA-β-gal activity." S1P -- SA-β-gal activity assay//BrdU assay "To test this hypothesis, we investigated whether SPHK1 knockdown-induced cellular senescence could be attenuated by inhibition of ceramide synthesis or exogenous supple- mentation with S1P during cell culture. BrdU incorporation assays showed that the proliferation impeded by shRNA- mediated silencing of SPHK1 could not be recovered by single treatment with S1P or fumonisin B1 (FB1), a ceramide synthesis inhibitor, and even by co-treatment with both , indicating the irreversibility of cellular senescence. In contrast, the elevated activity of SA-β-gal in SPHK1-silenced cells was reduced by simultaneous co-treatment with S1P and FB1, but not by single treatment with either one alone, suggesting that cellular senescence accelerated by SPHK1 depletion may result from increase in ceramide levels with concurrent decrease in S1P levels." -- -- -- -- Human L cellular senescence 30885289 Sen_G_0038 IGF1 3479 protein coding VSMC -- Aging Accelerate SA-β-gal activity assay We observed a strong induction of SA-β-gal activity following IGF-I stimulation in the confluent cells -- -- -- -- ROS-p53 -- Immunoblotting//SA-β-gal activity assay "We examined the involvement of ROS in the induction of cellular senescence by IGF-I.Treatment with an ROS scavenger, N-ace-tylcysteine (NAC), significantly suppressed the IGF-I-induced increase in SA-β-gal activity. Furthermore, NAC treatment also suppressed the induction of cH2AX, p53, and p21 proteins, indicating that ROS are involved in IGF-I-induced cellular senescence. Treatment with an alternative ROS scavenger,Trion, also produced similar results (data not shown). In addition,we used p53-/- MEFs to clarify the role of p53 in IGF-I-induced cellular senescence; in the absence of p53, IGF-I-induced SA-β-gal activation and p21 augmentation were inhibited." Human L cellular senescence 22877754 Sen_G_0039 SETD8 387893 protein coding PC-3 -- Aging Prevent Knockdown//Flow cytometry//SA-β-gal activity assay "In PC3 cells harboring SETD8-specific siRNAs,we detected a greater extent of β-gal-positive staining,cell proliferation arrest,nuclear area enlargement,G2/M arrest .Consistent with its known pro-proliferation role,overexpression of SETD8 in the absence of dox treatment elevated the rate of cell growth.In dox-treated cultures undergoing senescence,introduction of ectopic SETD8 lessened cellular senescence induction to a large extent, as evidenced by the reversal of β-gal-positive staining,cell proliferation arrest,and upregulation of senescence markers p21,IL-6,andIL-8." PPARc//p21 --//Downregulation qRT-PCR//SA-β-gal activity assay//CHIP//Flow cytometry "In support of its role as an upstream regulator, ectopic overexpression of PPARc led to an increase in the expression of SETD8 and H4K20me1, and conversely a decline in the p21 mRNA levels.To further demonstrate the role of PPARc in controlling senescence, we showed that overexpression of PPARc alleviated the extent of dox-induced cellular senescence, in terms of β-gal-positive staining , cell proliferation arrest, and SASP marker induction. Via ChIP-qPCR assay on the PPARc-overexpressing cells, we consequently found that the chromatin binding of PPARc increases on SETD8 promoter upon ectopic expression.Although RNAi-mediated abrogation SETD8 in cycling cell expectedly induced cellular senescence, based on the proportions of β-gal-positive cells and G2/M cells, simultaneous depletion of p21 notably suppressed these phenotypes. Further in line with this possible functional antagonism, concurrent depletion of p21 and SETD8 also lessened the progressive cell proliferation stall and nuclei enlargement, as compared with cells with down-regulated SETD8 only." -- -- -- -- Human HL cellular senescence 28514051 Sen_G_0040 YPEL3 83719 protein coding "MCF-7,U2OS" -- Aging Accelerate Colony formation assay//SA-β-gal activity assay "Using a colony formation assay,both cell lines showed considerably fewer colonies when expressing YPEL3 compared with cells not induced to express YPEL3. Growth suppression was also observed after shorter time periods of YPEL3 induction. Due to difficulties in the long-term growth of YPEL3 expressing cells, the inability to detect apoptosis, and observed morphologic changes, we examined the possibility that induction of YPEL3 would trigger cellular senescence.Two hallmarks of cellular senescence in human cells are the detection of increased acidic β-galactosidase activity and the appearance of foci within the nuclei of senescent cells . A clear increase (～38%) in cellular senescence was observed in U2OS/YPEL3-TetR cells exposed to tetracycline." p53 -- Dual-Luciferase reporter assay//Immunoblotting "The level of YPEL3 mRNA induction was significantly reduced in doxorubicintreated Hct116 cells lacking p53 .Cotransfection of the YPEL3-luciferase reporter with wild-type p53 in either Hct116?/?p53 or H1299 cells led to an increase in YPEL3 promoter activity .Having established that the YPEL3 promoter could be activated by p53 when cloned into a plasmid reporter construct, we next set out to test whether p53 protein associated with the YPEL3 promoter in vivo. p53 chromatin immunoprecipitation assays were performed with Hct116 cells undamaged (non-treated) or damaged with doxorubicin. As expected, p53 was shown to bindin vivoto the YPEL3 promoter in DNA-damaged Hct116 cells." -- -- -- -- Human HL cellular senescence 20388804 Sen_G_0041 IFNG 3458 protein coding HUVEC -- Aging Accelerate MTT assay//Flow cytometry//SA-β-gal activity assay "Cell proliferation decreased slightly with IFN-γ treatment in time- and dose-dependent manners. A decrease in cell proliferation by IFN-γ treatment for 3 days was statistically significant (p < 0.05). The induction of cellular senescence by prolonged treatment with IFN-γ was confirmed by an increase in SA-β-gal-positive cells in the treated cells compared to the control cells . IFN-γ treatment increased the G0/G1 cell population and decreased the S and G2/M cell population, suggesting that IFN-γinhibited cell proliferation through G0/G1 arrest in the cell cycle." p53 -- Knockdown//SA-β-gal activity assay//Western blot "Prolonged stimulation of IFN-γ in the p16-knockdown (p16sh) cells increased SA-β-gal activity staining to a level similar to that in control cells. In contrast, no increase in SA-β-gal staining was observed in the p53-knockdown (p53sh) cells following prolonged stimulation with IFN-γ. We reproducibly noticed an increase in the number of SA-β-gal-positive cells in p16-/-MEFs, but not in IFN-γ-treated p53-/-MEFs, after prolonged stimulation with IFN-γ. Therefore,these results suggest that IFN-γ-induced cellular senescence is mediated through a p53-dependent pathway.To confirm that p53 activation by IFN-γ treatment was mediated through DNA damage signaling,the ATM level was down-regulated using siRNAs against ATM,followed by treatment with IFN-γ for 6 days. Increases in p53 and phospho-p53 levels induced by IFN-γ treatment were repressed by knockdown of ATM." -- -- -- -- Human L cellular senescence 19071156 Sen_G_0042 HDAC4 9759 protein coding 2BS -- Aging Prevent Knockdown//Western blot//qRT-PCR//MTT assay//Flow cytometry//SA-β-gal activity assay "The overexpression and knockdown of HDAC4 were confirmed by western blot. Overexpression of HDAC4 resulted in only scattered senescence-associated β-galactosidase (SA-β-gal) activity, which is a biomarker for senescent cells. The formation of senescence-associated heterochromatin foci (SAHF), which is another hallmark of senescent cells, was not observed when HDAC4 was overexpressed. Continuous cell growth and a decrease in the proportion of 2BS cells in the G0/G1 phases were evident in HDAC4 overexpressing cells compared with corresponding empty control vector-infected cells. In contrast, HDAC4 knockdown induced more robust senescence phenotypes, which displayed an elevated SA-β-gal activity, prominent heterochromatin foci indistinguishable from those in senescent 2BS cells, pronounced cell growth arrest, and accelerated G1 cell cycle arrest compared with corresponding empty control vector-infected cells." SIRT1 -- Knockdown//RT-PCR//Western blot//IP "We first analysed the expression patterns of SIRT1 using western blot and RT-PCR subjected to such perturbations.The protein levels of SIRT1 were markedly enhanced in HDAC4-transfected HeLa cells compared with the vector-transfected cells. In addition, we silenced HDAC4 expression in HeLa cells. Not unexpectedly, there was a marked decrease in the SIRT1 protein level in these HDAC4 silenced cells.Moreover,the RT-PCR analysis demonstrated no alteration at the SIRT1 mRNA levels in either HDAC4-overexpressing or knockdown cells, indicating that HDAC4 might normally function to increase the SIRT1 protein levels, but not at the mRNA level.To gain further insight into the interplay between HDAC4 and SIRT1, cellular interactions between HDAC4 and SIRT1 were confirmed using immunoprecipitation (IP) assays." -- -- -- -- Human L cellular senescence 26414199 Sen_G_0043 ACLY 47 protein coding HDF -- Aging Prevent Knockdown//Cell morphological analysis//Cell proliferation assay//SA-β-gal activity assay ACLY knockdown cells showed apparent growth retardation and a typical senescent morphology (flat and enlarged in size). The proliferation assay and senescence-associated -β-galacto-sidase staining showed that knockdown of ACLY in HDF cells significantly reduced their proliferation rate. A large proportion of ACLY knockdown cells were positive for senescence-associated-β-galactosi-dase staining. -- -- -- -- p53-AMPK -- Knockdown//SA-β-gal activity assay//qRT-PCR//DAPI staining//Immunofluorescence//Pull-down assay "Initially, we silenced p53 in ACLY knockdown HDF cells, and found that additional knockdown of p53 completely abrogated ACLY silencing-induced cellular senescence. we observed an increased p53 protein level in ACLY knockdown HDF cells by confocalmicroscopy and increased mRNA levels of p53 downstream target genes by real-time quantitative PCR.The direct interaction of ACLY with AMPK a2 was confirmed by an in vitro glutathione S-transferase(GST) pulldown assay . Immunofluorescent staining of over-expressed AMPK and endogeneous ACLY showed that about 26% and 37% of the signal co-localized in HDF and HCT116 cells, respectively .An in vitro kinase assay using immunoprecipitated AMPK a2 showed an apparent reduction of AMPK activity by ACLY." Human L cellular senescence 25367309 Sen_G_0044 PIR 8544 protein coding "WM266-4,IGR-37" -- Aging Prevent Knockdown//Western blot//Colony formation assay//SA-β-gal activity assay//Cell morphological analysis "The growth rate was significantly reduced in both mela-noma cell lines stably expressing shPIR constructs,compared with that of cells bearing the empty pSICO-R. A colony formation assay was also performed to measure the growth rate at low density. The number of colonies formed by shPIR cells was significantly reduced in both cell lines.The shPIR-transduced cell lines exhibited changes in size and morphology compatible with cellular senescence (flattened and enlarged phenotype). We therefore performed a senescence-associated β galactosidase(SA-β-galactosidase) assay and found that PIR knockdown in WM266-4 cells resulted in an increase from 2.1% positivity in control cells to 41.3%, 16.6%, and 37.1% in shPIR#7, #16, and #19 transduced cells, respectively." -- -- -- -- -- -- -- -- Human L cellular senescence 21514450 Sen_G_0045 CUL4B 8450 protein coding "NHF,U2OS" -- Aging Prevent Knockdown//SA-β-gal activity assay//Western blot//BrdU assay "At day 5 post exposure to 1 Gy of ionizing radiation (IR), more than 9 % of normal human fibroblasts (NHFs) became senescent, as determined by assays of senescence-associated β–galactosidase (SA- β-gal) and BrdU incorporation. At this time point, there was a remarkable reduction in CUL4B at protein level. CUL4B was efficiently knocked down by RNA interference (RNAi). We observed that the percentage of senescent cells, as determined by SA- β-gal staining and BrdU incorporation, was significantly higher in shCUL4B cells than in shNeg cells after H2O2 treatment." -- -- -- -- p53-ROS Downregulation Knockdown//Flow cytometry//qRT-PCR//BrdU assay//SA-β-gal activity assay//Western blot "We used nutlin-3, which inhibits MDM2-mediated degradation of p53 and thus stabilizes p53, to activate p53 in NHFs and measured the level of ROS. Two days after nutlin-3 treatment the intracellular ROS level was higher in NHF-shCUL4B cells than in NHF-shNeg cells . Importantly, nutlin-3 failed to elevate the ROS level in CUL4B and p53 double knockdown cells (NHF-shCUL4B&shp53), while H2O2 treatment, as a positive control, caused an increase in the level of ROS in those cells. These results suggest that persistent activation of p53 alone may lead to an elevation in ROS level.We observed that while the percentage of senescent cells was higher in NHF-shCUL4B cells than in NHF-shNeg cells when treated with H2O2, the enhancement in senescence caused by CUL4B depletion was abolished in CUL4B and p53 double knockdown cells (NHF-shCUL4B & shp53), indicating that the enhancement in stress-induced senescence in CUL4B knockdown cells is dependent on p53. p53 activation, p53 transactivating activity and ROS production in response to H 2O2 treatment were significantly attenuated in PLOC-CUL4B cells ." Human L cellular senescence 25464270 Sen_G_0046 IGFBP5 3488 protein coding HUVEC -- Aging Accelerate RT-PCR//Western blot//SA-β-gal activity assay//MTT assay//Flow cytometry "The levels of IGFBP-5 mRNA and protein in old cells were down-regulated by gene silencing using IGFBP-5 micro-RNA lentivirus. Transduction with IGFBP-5 micro-RNA lentivirus caused 65% decrease in IGFBP-5 levels. Repression of IGFBP-5 levels in old cells caused morphological changes similar to young cells and a decrease in SA-β-gal activity. Treatment of young cells with rhIGFBP-5 decreased cell proliferation both time- and dose-dependently. Furthermore, prolonged treatment with rhIGFBP-5 increased SA-β-gal staining and caused a characteristically enlarged and flattened morphology.To confirm that rhIGFBP-5–treated cells entered senescence-related cell cycle arrest, we analyzed DNA content by flow cytometry with PI staining. Most cells stimulated with rhIGFBP-5 for 2 d were arrested in G1 phase, which is one of the typical phenotypes in cellular senescence." p53 -- Knockdown//Western blot//MTT assay "To further confirm that the p53 activation by IGFBP-5 might be mediated through DNA damage signaling, ATM level was down-regulated using siRNAs against ATM and the effects of IGFBP-5 overexpression were measured. Increases in p53 and phospho-p53 levels induced by IGFBP-5 up-regulation were repressed by knockdown of ATM. Furthermore, inhibition of cell proliferation induced by IGFBP-5 up-regulation was rescued by knockdown of ATM.These results suggested that IGFBP-5–induced cellular senescence is mediated by posttranslational modification of p53 such as phosphorylation and acetylation through DNA damage signaling." -- -- -- -- Human HL cellular senescence 17804819 Sen_G_0047 PLY 5601825 protein coding "BV-2,C6" -- Aging Accelerate SA-β-gal activity assay//Flow cytometry Senescence of glial cells (BV-2 and C6) was evident by SA-β-gal staining and morphological changes such as enlargement of the cell body and flat shape. Exposure to PLY for 24 h significantly increased the number of SA-β-gal positive cells compared with untreated control cells. PLY induced BV-2 cell cycle arrest in the G0/G1 phase during the 24 h exposure. -- -- -- -- MAPK-NF-κB Activation SA-β-gal activity assay//Western blot//Co-IP "Phosphorylation of ERK1/2, p38 MAPK, and JNK was increased in PLY-treated BV-2 cells .NAC inhibited MAPK activities by pretreatment with for 2 h followed by incubation with PLY for 12 h . Increased expression of PAI-1 in PLY-induced BV-2 cells was significantly decreased by the MAPK inhibitors. In addition, treatment with MAPK inhibitors significantly reduced the number of SA-β-gal positive cells. PLY treatment enhanced the expression of p65 subunit of NF-kB in a time-dependent manner.We also investigated whether the interaction between SIRT-1 and p65 proteins is affected by PLY treatment, given the SIRT-1 interacts with p65 in modulating cell fate in response to various signaling events. The interaction was confirmed by a co-immunoprecipitation assay. SIRT-1 and p65 physically interacted with each other at 4 h or 24 h in PLY-treate BV-2 microglial cells . Altogether, these results suggest that PLY induces cellular senescence via the SIRT-1 and NF-kB interaction in BV-2 cells.All the MAPK pathway inhibitors differentially reduced the expression of p-SIRT-1 and NF-kB enhanced by PLY." Human L cellular senescence 28232188 Sen_G_0048 MCAM 4162 protein coding "CD1462,CD146+" -- Aging Prevent SA-β-gal activity assay//qRT-PCR//TRAP assay//Immunoblotting//Knockdown "Specifically,the growth of CD1462cells ceased at P12, whereas CD146+ cells continued to grow until P15. In qPCR analysis, CD146+ cells showed high expression levels of stemness genes compared with those observed in CD1462 cells . Similarly, telomerase activities of CD146+ cells were significantly higher than those of CD1462 cells . Western blot analysis indicated that CD1462 cells showed enhanced expression of pho-p53 and p16 relative to that observed in CD146+ cells, as well as significantly augmented SA-β-gal activity and cell areas .Cells with silenced CD146 expression exhibited a reduced duration of proliferation during the culture period and lower growth rates, compared with the respective measures made in na?ve cells or scrambled siRNA-transfected cells. SA-β-gal activities and cell areas were significantly increased in CD146 siRNA transfectants." -- -- -- -- -- -- -- -- Human L cellular senescence 26941359 Sen_G_0049 TFAP4 7023 protein coding RPE -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis "The morphological changes in AP4-expressing cells, including enlarged nuclei and flattened cytoplasm, were characteristic of cellular senescence .When AP4 expression was induced by adding DOX to the media, the proliferation profiles of RPE-AP4 cells started to differ on Day 12 ARC from the cells without receiving DOX. AP4-expressing cells stopped proliferating, and the status of these cells remained constant for at least additional 12 days.To quantify the extent of AP4-induced senescence, the number of blue SA-β-gal-positive cells was counted.The results showed that substantially more AP4-expressing cells than control cells were SA-β-gal positive." -- -- -- -- p14-p53 Activation qRT-PCR//Immunofluorescence "Immunofluorescence microscopy was used to investigate the expression of p14ARF, p53,and Rb proteins in AP4-induced senescent RPE cells. p53 protein was expressed primarily in the large senescent AP4-expressing cells but not in DN-AP4-expressing cells.A similar expression pattern was observed in p14ARF, which appeared as small speckles distributed throughout the nucleoplasm but not the nucleolus of AP4-expressing cells .To determine whether AP4 can regulate p53 at the transcriptional level, the levels of p53 mRNA were quantified using qRT-PCR. The p53 gene has two promoters, P1 and P2 ,which are the transcription starting sites of the p53 gene and produce transcripts of different lengths." Human HL cellular senescence 26439195 Sen_G_0050 AGR2 10551 protein coding "LNCaP,DU 145,PC-3" -- Aging Prevent MTT assay//qPCR//Western blot//Flow cytometry//SA-β-gal activity assay "Forced expression of AGR2 in LNCaP or DU145 cells for 72 h resulted in a marginal increase in the cell viability as measured by MTT assays. V alidation of the AGR2 expressions at mRNA and protein levels was performed by quantitative PCR and western blot assays. AGR2 expressions were markedly increased in the transfected LNCaP or DU145 cells compared with the vector-transfected cells. Reduced endogenous AGR2 in PC-3 cells led to decreased cell viability, whereas no decreases were observed in cells transfected by control oligonucleotides.Transfection of LNCaP, PC-3 and DU145 cells with AGR2i led to significantly increase in the percentage of cells in G /G1phase compared with the control cells." -- -- -- -- -- -- -- -- Human HL cellular senescence 22467239 Sen_G_0051 PLK1 5347 protein coding "HDF,HUVEC" -- Aging Prevent Western blot//SA-β-gal activity assay//Cell counting//BrdU assay//Cell morphological analysis " Replicative senescence in cells was confirmed by increases in SA-β-gal staining and the levels of p53, p21, and p16 proteins as well as enlarged, flattened cell morphology . The expression levels of PLK1 mRNA and protein decreased in old cells compared with young cells. PLK1 immunoreactivity was stronger in young cells than in old cells and PLK1 was localized in the nucleus or chromosomes of young cells under mitosis . Furthermore, the level of PLK1 protein also decreased during adriamycin-induced premature cellular senescence.Following transduction of old cells with recombinant PLK1 adenovirus, upregulation of PLK1 protein levels was confirmed by Western blot analysis. PLK1 overexpression in old cells increased cell proliferation , BrdU incorporation, and PDs . PLK1 upregulation decreased SA-β-gal staining in a dose-dependent manner." -- -- -- -- p53 -- Knockdown//Western blot//Cell counting//SA-β-gal activity assay "Downregulation of p53, p21, and PLK1 was confirmed by Western blotting. However, we could hardly detect the p16 protein in our experimental condition because young cells were known to express very low level of p16 protein in normal condition. Instead, we confirmed the knockdown of p16 by transfecting old cells with the p16 shRNA vector since old cells show an increase in the p16 level. Although p16 shRNA–treated cells showed senescence phenotypes induced by PLK1 knockdown, such as decreased cell proliferation and increased SA-β-gal staining, p53 shRNA–treated cells did not." Human HL cellular senescence 23525475 Sen_G_0052 HJURP 55355 protein coding "HDF,HUVEC" -- Aging Prevent SA-β-gal activity assay//DAPI staining//Flow cytometry//RT-PCR//Western blot//Cell counting "Ectopic expression of HJURP in senescent cells increased cell proliferation in a time-dependent manner and resulted in morphological changes similar to young cells and a decrease in SA-β-gal staining activity.In addition, the upregulation of HJURP decreased the cell population in the G1 phase of the cell cycle, which is increased in old cells and cells with control vector and elevated the G2/M cell populations, which is declined in old cells and control vector. Because upregulation of HJURP levels partially reversed cellular senescence, we examined the effects of HJURP knockdown on senescence phenotypes in young HDFs and HUVECs by transfection of HJURP siRNAs. HJURP knockdown was confirmed by RT-PCR and Western blotting . Although the levels of pA TM, p53, and p21 were elevated, the levels of pRb and CENP-A were repressed by HJURP knockdown." -- -- -- -- p53 -- Knockdown//Western blot//RT-PCR//SA-β-gal activity assay "To determine the pathway involved in the regulation of cellular senescence by HJURP reduction, we knocked down p53 or p16 levels in young cells and measured the effects of HJURP downregulation on cellular senescence. The levels of HJURP, p53, and p16 were confirmed by RT-PCR and Western blotting. HJURP knockdown decreased cell proliferation in p16 siRNA cells but not in p53 siRNA cells. Similarly, depletion of HJURP increased SA-β-gal staining activity in p16 siRNA cells but not in p53 siRNA cells." Human HL cellular senescence 23292286 Sen_G_0053 ERRFI1 54206 protein coding 2BS -- Aging Accelerate Western blot//BrdU assay//SA-β-gal activity assay//SAHF "The Mig-6 overexpression was confirmed by Western blot analysis in the pLVX-Mig-6 cells. The results show that the pLVX-Mig-6 cells overexpressing Mig-6 showed reduced DNA synthesis.The pLVX-Mig-6 cells displayed the flat morphology, stained positively for senescence-associated-β-galactosidase (SA- β-gal) and formed senescence-associated heterochromatin foci (SAHF) 12 days after lentiviral infection (8 days post-selection). Moreover, molecular markers of senescence, such as increased p53, p21, p16 and decreased p-Rb levels, were detected after Mig-6 overexpression." FOXO3a -- Knockdown//RT-PCR//Immunobloting//Western blot "Ly294002 treatment dramatically increased Mig-6 expression in young cells. In addition, RNA interference was used to deplete FOXO3A protein in RasV12-induced senescent cells. The amount of FOXO3A protein in the cells was reduced by at least 80% by a specific shRNA as compared with the control shRNA. FOXO3A knockdown reduced Mig-6 mRNA and protein levels in these senescent cells. Results showed that Mig-6 or FOXO3A knockdown partially restored the response of Ras-induced senescence to EGF , which was suggested by the Akt phosphorylation levels." EGFR-ERbB Downregulation Western blot//SA-β-gal activity assay "Western blot analysis showed that levels of the phosphorylated EGFR (p-EGFR) were evidently decreased,but its total expression levels were not obviously altered in the senescent cells compared with those in the young cells.Deletion of ErbB-2-binding domain did not affect the phosphorylation levels of p-EGFR,p-Akt (phosphorylated Akt) and p-Erk (phosphory-lated Erk), and greatly reduced the ability of Mig-6 to enhance SA-β-Gal staining compared with the wild-type Mig-6. The result showed that as pcDNA-EGFR amount increased, SA-β-Gal activity decreased with a dosedependence,suggesting that EGFR can an-tagonise of Mig-6 induction effect of cellular senescence.AG1478 (but not AG9)treatment resulted in the decreased EGFR phosphorylation and the increase of SA-β-Gal activity, suggesting that the suppression of ErbB signalling only can elicit a cellular senescence. Subsequently, the EGFR inhibitor AG1478 treatment resulted in the increased Mig-6 expression , but not reduced Mig-6 expression." Human HL cellular senescence 23746120 Sen_G_0054 ING1 3621 protein coding IMR-90 -- Aging Accelerate Western blot//BrdU assay//SA-β-gal activity assay//Cell morphological analysis "Enforced expression of ING1 caused a dramatic reduction in proliferation, as shown by a decrease in thymidine incorporation or in the number of BrdU-positive cells.The inverse correlation between ING1 expression and BrdU incorporation could also be observed in individual cells by immunofluorescence. Eighty-five percent of AU5-positive cells were BrdU-negative, while 65% of AU5-negative cells or vector-infected cells were BrdU-negative, confirming the antiproliferative effect of ectopic ING1.ING1-expressing fibroblasts also displayed distinctive markers of cellular senescence,such as SA-Beta Gal activity , and flat enlarged morphology." -- -- -- -- p53 -- Western blot//SA-β-gal activity assay "To investigate the functional connection between p33ING1 and p53 in the context of senescence, we inactivated the p53 pathway in these cells, using the E6 oncoprotein from human papilloma virus. Ectopic expression of p33ING1 failed to induce a significant cell-cycle arrest in fibroblasts expressing E6 .Similarly, the induction of the senescence marker SA-β-Gal by p33ING1 was largely abolished in E6-expressing cells. In agreement with previous reports, inactivation of the p53 pathway was not sufficient to bypass senescence by RasV12 in these cells, which requires additional inactivation of the Rb pathway. Interestingly, ING1 increased the level of p21CIP1, a p53 target associated with senescence. The induction of p21CIP1 by ING1 was lost in E6-expressing cells ." Human HL cellular senescence 21078114 Sen_G_0055 SIRT6 51548 protein coding HepG2 -- Aging Prevent SA-β-gal activity assay Inhibiting the upregulation of SIRT6 enabled TGF-β1?H2O2?HOCl to efficiently induce cellular senescence. -- -- -- -- ERK//Smad//p38 MAPK --//--//-- SA-β-gal activity assay "Consistent with the promoting effect of Smad and p38 MAPK pathways on the expression of SIRT6, inhibiting each of these pathways enabled TGF-β1?H2O2?HOCl to induce the cellular senescence of HCC cells. Transforming growth factor-β1?H2O2?HOCl could induce sustained and enhanced activation of the ERK pathway.Consistently, TGF-β1?H2O2?HOCl could efficiently induce cellular senescence, if the ERK pathway was inhibited. In this situation, the effect of TGF-b1?H2O2?HOCl on cellular senescence was also attenuated by inhibiting the NF-jB,Smad, and p38 MAPK pathways, which was consistent with the effect of suppressing the upregulation of SIRT6. However, if SIRT6 expression was suppressed by shRNA, TGF-β1?H2O2?HOCl could similarly induce cellularsenescence, even though the ERK pathway was activated ." Human L cellular senescence 25683165 Sen_G_0056 MYC 4609 protein coding RPE -- Aging Accelerate SA-β-gal activity assay//Knockdown "The SA-β-gal activity assay was performed on these two cell clones with or without 4-OHT treatment on Day 24. With the addition of 4-OHT, the average number of SA-β- gal-positive cells per unit area was increased in both the cells with LL-c-Myc (clone 4) and ML-c-Myc (clone 3). In order to determine whether long-term post-confluent cells entered into senescence rather than cellular quiescence, we replated the confluent RPE LL-c-Myc cells (clone 4) treated with OHT for 24?days, and measured their SA-β-gal activity at different time points. The percentage of β-gal-positive cells after 2 and 6?days was 85.6 and 87.3%, respectively, suggesting that the replated cells were not proliferating, and the confluent cells were substantially senescent rather than quiescent. Collectively, we concluded that low level of c-Myc expression (LL-c-Myc) induced cellular senescence rather than quiescence in post-confluent RPE cells during long-term culture.After infecting with lentivirus expressing shRNA, all the cells expressing high level of c-Myc (HL-c-Myc) eventually died after 8?days, while HL-c-Myc cells infected with scrambled lentiviral shRNA control could survive for 12?days. For ML-c-Myc cells,it took 24?days for the cells infected with scrambled lentiviral shRNA to die completely." AP4//p53 --//-- Knockdown//Western blot "The expression of AP4 increased evidently in the samples that incubated with 4-OHT for the initial 14?h, while c-Myc-ER level was relatively high. Both AP4 and c-Myc-ER protein were subsequently decreased during longer incubation, which might probably due to protein degradation. After normalized to the signal of actin, the expression of AP4 increased sevenfold during the first 14?h upon the activation of c-Myc,suggesting that RPE c-Myc-ER clones were functional and could be used for further studies.Western blot analysis showed that differential c-Myc expression in the cell clones induced a corresponding level of AP4 protein.In the cells with LL-c-Myc, the expression of p53 decreased more with a larger decrease in AP4 level, indicating that p53 expression was regulated by AP4, which is consistent with our previously finding .In this study, knockdown of AP4 expression brings about a decrease in p53 expression, but the decrease in the expression of p53 was weak in RPE cells when c-Myc level is higher." -- -- -- -- Human L cellular senescence 29188535 Sen_G_0057 CDKN2A 1029 protein coding Lymphoma cell line -- Acute leukemia Accelerate Knockdown//SA-β-gal activity assay Acute knockdown of p19ARF expression by shRNA resulted in decreased SA-beta-galactosidase activity. -- -- -- -- -- -- -- -- Human HL cellular senescence 25784651 Sen_G_0058 ATF7IP 55729 protein coding IMR-90 -- Aging Prevent EdU assay//SA-β-gal activity assay//DAPI staining//Knockdown MCAF1 knockdown severely impaired cell proliferation and reduced incorporation of a thymidine analog EdU;MCAF1 knockdown cells were positive for SA-β-gal activity.DAPI staining showed that approximately 3 % of MCAF1 knockdown cells displayed nuclear foci that resemble SAHF. -- -- -- -- Rb -- Western blot "We also found that the cdk inhibitors p16 and p21 were upregulated at both mRNA and protein levels, and RB protein was dephosphorylated in MCAF1 knockdown cells." Human HL cellular senescence 23935871 Sen_G_0059 TNFSF13 8741 protein coding SW480 -- Colorectal cancer Prevent SA-β-gal activity assay "These results indicated cellular β-galactosidase activity, a symbol of cell ageing, had enhanced compare with that of no pGshAPRIL-transfection." -- -- -- -- HSPG -- Western blot//SA-β-gal activity assay "By contrast, high levels of SA-β-gal activity were exhibited in pGshAPRIL-transfected plus TACI-Fctreated groups or those transfected with pGshAPRIL (P< 0.01). SA-β-gal activity was shown to increase in groups with pGshAPRIL and heparin combined treatment and so did in pGshAPRIL, TACI-Fc add heparin treatment groups (P<0.05)." Human L cellular senescence 19466596 Sen_G_0060 GNG11 2791 protein coding Fibroblast -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis "We transfected plasmid encoding GNG11 (pG11-sense),they stopped growing 1 week about after transfection, exhibited an enlarged and flat cell shape, and were clearly stained with senescence-associated β-galactosidase." -- -- -- -- ERK1/2 Activation Western blot "Both ERK1/2 were significantly activated 5 days after transfection, whereas little or no activated forms of p38 and JNK were detected." Human L cellular senescence 17092487 Sen_G_0061 MDH1 4190 protein coding HDF -- Aging Prevent Knockdown//SA-β-gal activity assay//Cell morphological analysis "We found that about 60 % of SA-β-Gal-positive cells appeared among MDH1 knockdown cells, but only 10 % among control cells. shMDH1-infected young HDFs also became flattene and enlarged；cell numbers in cultures of MDH1 knockdown HDFs were decreased by about 50 % compared to control cells plated at the same density and cultured for the same duration ." SIRT1 -- Knockdown//Western blot//RT-PCR SIRT1 RNA levels were decreased in MDH1 knockdown cells. -- -- -- -- Human L cellular senescence 22971926 Sen_G_0062 SMARCB1 6598 protein coding Lac-hSNF5 -- Malignant Rhabdoid Tumor Accelerate BrdU assay//DAPI staining//SA-β-gal activity assay//Flow cytometry "We found that hSNF5 induction dramatically impaired cell proliferation, even when cultured in the presence of 1 % serum . In contrast, addition of IPTG had no effect on Lac-empty control cells lacking the hSNF5 gene. Similar results were obtained with independently isolated Lac-hSNF5 cell lines (data not shown). We also assessed the effect of hSNF5 on the colony-forming capacity of Lac-hSNF5 cells, and found that hSNF5 expression dramatically reduced the number of colonies formed, after seeding these cells at a very low density.We next determined which stage of the cell cycle is affected by hSNF5.Expression of hSNF5 leads to impaired DNA synthesis, indicated by the strongly reduced incorporation of BrdUrd. FACS analysis revealed that the hSNF5-expressing cells accumulate in G1, suggesting that the cell cycle block affects entry into S-phase." -- -- -- -- p16-pRb -- Western blot "Western immunoblotting revealed a strong up-regulation of p16INK4aprotein levels following hSNF5 expression, whereas no increase or even a small reduction of p14ARFwas observe.Because p16INK4a is a specific inhibitor of the Rb-regulating cyclin D1-CDK4 complex, we determined the phosphorylation status of pRb in Lac-hSNF5 cells. In support of a role of hSNF5." Human L cellular senescence 14604992 Sen_G_0063 WWP1 11059 protein coding 2BS -- Aging Prevent SA-β-gal activity assay//Knockdown//Flow cytometry "Senescence markers for WWP1-overexpressing and WWP1 shRNA-infected cells were then monitored at the same time points as their corresponding control cells. The WWP1 shRNA-transfected 2BS cells displayed enlargement, flattening, and the accumulation of vacuolated cytoplasmic inclusions. However, no significant morphological changes were observed in the WWP1-transfected cells, which were similar to young 2BS cells. Most of the WWP1 shRNA-transfected cells showed strong blue SA-β-gal staining similar to senescent cells . In contrast, only scattered SA-β-gal-positive cells were found in WWP1-transfected cells compared with the corresponding control cells. 2BS cells overexpressing WWP1 showed increased S and reduced G1 compartments. It was obvious that WWP1 shRNA-infected 2BS cells entered G1cell cycle arrest." -- -- -- -- p27 -- Knockdown//RT-PCR//Western blot "The protein levels of p27Kip1were markedly depressed in WWP1-transfected HeLa cells as compared with the vector-transfected cells, whereas the protein levels of p16INK4a and PTEN were invariable. In addition, we silenced WWP1 in HeLa cells. Similarly, there was no detectable change in the protein levels of p16INK4a or PTEN, but a marked increase in the p27Kip1 protein level was found. Moreover, the RT-PCR analysis demonstrated no alteration in p27Kip1mRNA levels in both WWP1-overexpressing and knockdown cells, which indicated that WWP1 might normally decrease the p27Kip1 protein level but not the mRNA level. Similar results were observed in 2BS cells." Human HL cellular senescence 21795702 Sen_G_0064 ATP6V0A2 23545 protein coding TIG-1 -- Aging Prevent RT-PCR//EdU assay//SA-β-gal activity assay "We evaluated the functional role of ATP6V A2 in cellular senescence of the TIG-1 cells. The mRNA expression of senescence markers p21 and p16, and protein expression of senescence markers p21, p16, phospho p38 and γH2AX significantly increased in ATP6V A2-silenced young and old TIG-1 cells. When DNA replication was assayed by examining 5-ethynyl-2′-deoxyuridine (EdU) incorporation in a pulse-label experiment, there was a steady decrease in EdU incorporation in ATP6V A2-silenced young TIG-1 cells and old TIG-1 cells. Growth was greatly reduced in ATP6V A2-silenced young TIG-1 cells and old TIG-1 cells. The activity of senescence-associated -β-galactosidase (SA-β-Gal) was augmented in ATP6V A2-silenced young TIG-1 cells and old TIG-1 cells." -- -- -- -- -- -- -- -- Human L cellular senescence 26611489 Sen_G_0065 KIF2C 11004 protein coding "HDF,HUVEC" -- Aging Prevent SA-β-gal activity assay//Western blot//RT-PCR "We tried to investigate the role of MCAK/Kif2C in cellular senescence.Cellular senescence of HDFs and HUVECs was confirmed by increases in SA-β-gal activity and p53 levels .Decreased expression levels of MCAK/Kif2C were shown in old cells by RT-PCR, real-time PCR, and Western blotting." -- -- -- -- p53 -- Knockdown//RT-PCR//Western blot "Knockdown of p53, p16, and MCAK/Kif2C were confirmed by RT-PCR and Western blotting. A decrease in cell proliferation and an increase in SA-β-gal activity by MCAK/Kif2C knockdown were not observed in p53 siRNA cells, but were evident in p16 siRNA cells. Senescent-like cell morphology was also shown in p16 siRNA cells, but not p53 siRNA cells . These results support the view that premature cellular senescence induced by MCAK/Kif2C knockdown might be regulated via a p53-dependent pathway." Human HL cellular senescence 23098759 Sen_G_0066 CREG1 8804 protein coding Fibroblast -- Li-Fraumeni Syndrome Accelerate Western blot//MTT assay//Cell proliferation assay "The expression level of CREG1 fits the criteria of senescence-associated gene, decreased during immortalization and increased in replicative and 5-aza-dC-induced senescence justifying further investigation of the role of CREG1 in cellular senescence.We found that CREG1 overexpression decreased cell growth in immortal LFS cells when compared with pIRES empty vector transfected cells demonstrated by both MTT and cell counting proliferation assays." p16 Upregulation Cell cycle analysis//SA-β-gal activity assay//Western blot//Cell morphological analysis "Cell cycle analysis showed an increase in the G0-G1 cell population and a decrease of S-phase population in cells cotransfected with CREG1 and p16INK4a compared to cells transfected with either CREG1 or p16INK4a alone.In p16-transfected cells and more so in cells cotransfected with CREG1 and p16INK4a, we observed cell characteristics indicative of senescence, a large and flat morphology with an increase of SA-β-galactosidase staining.We found that cells coexpressing CREG1 and p16INK4a showed a marked decline in protein levels of cyclin A and B ." -- -- -- -- Human HL cellular senescence 21263217 Sen_G_0067 TOPBP1 11073 protein coding "MEF,3T3" -- Aging Prevent SA-β-gal activity assay//DAPI staining//Flow cytometry "TopBP1-ablated cells underwent morphological changes including enlargement and flattening of the cells on the culture dishes.TopBP1-ablated cells were positive with respect to SA-β-gal staining,which detects senescent cells.FACS analysis of TopBP1-ablated cells indicates a reduction of G1-phase cells containing 2N DNA and a reduction of S-phase cells as well as an increase of G2/M-phase cells containing 4N DNA content, relative to wild-type cells." -- -- -- -- -- -- -- -- Human L cellular senescence 21149450 Sen_G_0068 GRSF1 2926 protein coding WI-38 -- Aging Prevent Immunofluorescence//Western blot//SA-β-gal activity assay "Immunofluorescence analysis confirmed that GRSF1 levels declined in senescent cells and Western blot analysis further established that GRSF1 levels decreased early in senescence SA-β-gal analysis revealed greater numbers of positive (blue) senescent cells after GRSF1 depletion, supporting the notion that lowering GRSF1 promoted cell senescence." IL-6 -- qRT-PCR//ELISA "The levels of IL6 mRNA and secreted IL6 were significantly higher in GRSF1-silenced WI-38 fibroblasts, as determined by RT-qPCR analysis and AlphaLISA， respectively." -- -- -- -- Human L cellular senescence 30086537 Sen_G_0069 PRKAA1 5562 protein coding BEAS-2B Lung Chronic obstructive pulmonary disease Prevent qRT-PCR//Cytokine assay//Immunocytochemistry//Western blot "Transfection of AMPKα1/α2 siRNA increased the mRNA of p16, p21 and p66shc, but reduced klotho gene expression in BEAS-2B cells.We also determine the expression of p16 and p21, markers of cellular senescence, in lung tissues using Western blot and immunohistochemistry." SOD2//SIRT3 Upregulation//Upregulation Immunocytochemistry//Western blot "In response to elastase, the abundance of SOD2 and SIRT3 proteins was significantly reduced as compared to saline control.The expression of SOD2 and SIRT3 proteins was augmented by prophylactic treatment of metformin, whereas Compound C further caused reduction of SOD2 and SIRT3 proteins in emphysematous lungs." -- -- -- -- Human L cellular senescence 28186975 Sen_G_0070 SLC25A5 292 protein coding "U2OS,MCF-7" -- Aging Prevent BrdU assay//Knockdown//Cell morphological analysis//SA-β-gal activity assay "Down-regulation of ANT2 by siRNA led to a decreased BrdU incorporation indicating inhibition of cell proliferation .Further decrease in BrdU incorporation was observed after the combined treatment. Although short term knock-down of ANT2 did not enhance senescence associated β-galactosidase staining, enhanced vacuolization, typical of senescent cells, was observed in cells with combined treatment." NF1/Smad -- IP//CHIP//qRT-PCR//Flow cytometry Induction of the NF1/Smad complexes and binding of NF1 to the ANT2 NF1-dependent repressor elements followed the same time course as ANT2 repression in U2OS cells. TGFβ -- qRT-PCR//Flow cytometry These events are also closely correlated with the expression and secretion of TGF-β suggesting that formation of the NF1/Smad complexes is initiated through TGF-β signaling. Human L cellular senescence 25220407 Sen_G_0071 TP53 7157 protein coding "MCF-7,A549,HCT116" -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis//Cell proliferation assay "Consistent with these observations, we found that overexpression of human p53β increased markers of cellular senescence in a variety of human tumor cell lines (MCF-7, A549, HCT116), including enlarged cell size, decreased cell proliferation, and elevated senescence-associated β-galactosidase (SA-β-gal) activity." -- -- -- -- hSMG-1-RPL26-SRSF7 -- SA-β-gal activity assay//Knockdown "Further, modulation of the hSMG-1-RPL26-SRSF7 pathway required for IR-induced p53β alternative splicing also affected cellular senescence markers as well. Knock-down of hSMG-1, which activates this splicing pathway, increased the percentage of SA-βgalactosidase positive cells and knock-down of SRSF7, a positive splicing factor for p53β, suppressed IR-induced SA-β-galactosidase induction." Human HL cellular senescence 28288992 Sen_G_0072 SRSF3 6428 protein coding Fibroblast -- Aging Prevent SA-β-gal activity assay//Knockdown "The cells transfected with siSRSF3 no. 1 and siSRSF3 no. 2, but not those with a control siRNA (siControl), underwent a senescent growth arrest rapidly (within 7 days) with concomitant induction of the SA-β-gal activity." tp53 Downregulation RT-PCR//qRT-PCR//Western blot//Knockdown "Knockdown of SRSF3 also increased p53b protein level, reminiscent of its accumulation in replicative senescence. When the alternative splicing patterns of TP53 were examined by conventional RT-PCR and qRT-PCR,mRNA levels of p53b were remarkably upregulated upon SRSF3 knockdown, while the levels of fulllength p53 mRNA were moderately decreased." -- -- -- -- Human HL cellular senescence 22777358 Sen_G_0073 EZH2 2146 protein coding "SK-Mel-119,UCD-Mel-N,SK-Mel-173" -- Melanocytic nevi Prevent Immunofluorescence//Flow cytometry//SA-β-gal activity assay//qRT-PCR//SAHF//Cell morphological analysis//Knockdown "Growth of EZH2 siRNA cells was significantly decreased compared to control-transfected cells. EZH2 siRNA cells displayed a flattened, enlarged morphology and expressed SA-β-Gal. In addition to SA-β-Gal expression, additional senescence markers following extended EZH2 depletion (6 days) include SAHFs and the heterochromatin-associated H3K9me3 modification as well as an increase in cell size, as evaluated by tubulin immunofluorescence. Flow cytometric analysis demonstrated that the proportion of cells in the S phase decreased in EZH2 siRNA cells with a concomitant increase of cells in the G1 phase, a significant difference in all 3 cell lines tested. In SK-Mel-173 cells, we also noticed a slight increase in the proportion of cells in the G2 phase." p21 -- Western blot "However,depletion of EZH2 elevated expression of p21 in 5/6 cell lines." -- -- -- -- Human L cellular senescence 21383005 Sen_G_0074 TAGLN 6876 protein coding HepG2 -- Aging Accelerate Colony formation assay//SA-β-gal activity assay "SM22a overexpression significantly induced the inhibition of cell growth of HepG2 cells. Conversely, when SM22a-overexpressing cells were transfected with siRNA, which suppresses the expression of SM22a, the cell growth was partially restored to that of control cells.These results suggest that SM22a is closely involved in regulation of cell growth and senescence." MT-1G Upregulation RT-PCR//Western blot "In two selected SM22a-overexpressing clones, the levels of MT-1G were also elevated." p16-pRb Activation RT-PCR//Western blot "SM22a overexpression resulted in a significant elevation of p16INK4a when analyzed by RT-PCR and Western blot. In wild-type HepG2 cells, serine 780 and 807/811 residues of pRB were highly phosphorylated. However, SM22a overexpression in these cells significantly inhibited the phosphorylation of serine 780 and 807/811 residues in pRB, which may prevent the detachment of E2F from pRB and thus suppress cell proliferation. Phosphorylation of serine 608 residue in pRB was also significantly inhibited. On the other hand, when SM22a-overexpressing cells were transfected again with siRNA to suppress SM22a expression, serine 608, 780, and 807/811 residues in pRB were re-phosphorylated, thereby restoring cell growth to a certain level." Human L cellular senescence 20705054 Sen_G_0075 SLC13A3 64849 protein coding WI-38 -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis "In addition, the volume of WI38/hNaDC3 cells in PD37 showed hypertrophy compared to that of control and empty vector groups.The SA-beta-gal (a biomarker of cell senescence) staining results showed that the percentage of staining positive cells in WI38/hNaDC3 group (98.5± 5.6%) was significantly greater than that in the WI38/con (22.3 ± 2.4%) and WI38/neo group (23.4 ±2.18%) (P< 0.05) in 37 PD." -- -- -- -- SIRT1 Downregulation Fluorescence microscopy "To determine whether NaDC3 induces aging through inhibiting SIRT1 pathway, SIRT1 deacetylase activity was detected by fluorescent assay. The results showed that the activity was down-regulated in the WI38/hNaDC3 group (9256 ± 675), compared with that in the control (20,569 ± 1035) and the WI38/neo groups (18,972 ±1158) (P < 0.05)." Human L cellular senescence 20813124 Sen_G_0076 PRDX3 10935 protein coding "HTR8,Human primary trophoblast" -- Intrahepatic cholestasis of pregnancy Prevent ROS assay//SA-β-gal activity assay//Flow cytometry "As expected, knockdown of PRDX3 significantly induced mitochondrial dysfunction, as indicated by overproduction of ROS, loss of mitochondrial membrane potential and decline in ATP content .Subsequently, the cell cycle distributions of these cell strains were analysed with flow cytometer. Compared to the sh-scr cells, shPRDX3-1# and shPRDX3-2# cells exhibited prolonged G0/G1 phase, indicative of growth arrest.In our study, PRDX3-knockdown cells showed a significantly higher number of SA-β-gal stained cells compared with sh-scr control cells at basal level." p16//p21//p38 --//--//-- qRT-PCR//Western blot//HC assay "QRT-PCR assay revealed that mRNA levels of p21WAF1/CIP and p16INK4A were increased in PRDX3-knockdown cells compared with sh-scr cells.Immunoblot assay also confirmed the corresponding change of protein levels of p21WAF1/CIP and p16INK4A in PRDX3-knockdown cells,and in ICP placentas by immunoblot and IHC assays.Indeed, we observed elevated p38-MAPK phosphorylation in PRDX3-knockdown cells." -- -- -- -- Human HL cellular senescence 27958341 Sen_G_0077 UBE2E3 10477 protein coding RPE-1 -- Aging Prevent ELISA//SA-β-gal activity assay//EdU assay//Western blot//Flow cytometry "Depletion of UBE2E3 led to a loss of proliferation, as indicated by a failure of siUBE2E3 cells to incorporate 5- ethynyl-2 -deoxyuridine (Edu), a marker of newly-synthesized DNA ,an increase in cell size,a robust expression of senescence-associated β-galactosidase , a decrease in intracellular HMGB1 content,redistribution of lamin B from its primary localization at the nuclear envelope to a punctate, nucleoplasmic distribution and a concomitant decrease in lamin B1 mRNA expression,increased transcription of the tumor suppressor p16INK4a, and increased secretion of the pro-inflammatory cytokine IL-6 ." -- -- -- -- p53-p21 -- SA-β-gal activity assay//EdU assay "Using a combination of co-knockdowns and measuring both SA-β-Gal expression as well as Edu incorporation in the same cells, 4 days after siRNA administration, we found that silencing the expression of either p53 or p21CIP1/WAF1 mitigated the siUBE2E3- induced SA-β-gal expression whereas silencing either p21CIP1/WAF1 or p16INK4a mitigated the cell cycle exit." Human L cellular senescence 29879550 Sen_G_0078 FAHD1 81889 protein coding HUVEC -- Aging Prevent SA-β-gal activity assay//Western blot//Knockdown "Furthermore,depletion of FAHD1 induced premature senescence in HUVEC, as shown by an increased percentage of cells staining positive for senescence-associated β galactosidase (SA-β-gal) .We found that the levels of both senescence marker proteins were significantly down-regulated in FAHD1-depleted cells.These findings suggest that FAHD1 is required for cell proliferation in human endothelial cells and its inactivation induces premature senescence." p53//p21/WAF1 --//-- Western blot "Of note, we observed slightly increased p53 protein levels in FAHD1-depleted cells, which were accompanied by a significant increase in the level of the cdk inhibitor p21Cip1/Waf-1, which is known to contribute to the senescence-associated proliferation arrest." -- -- -- -- Human L cellular senescence 28286170 Sen_G_0079 KCNH7 90134 protein coding A375 -- Melanoma Accelerate Western blot//Flow cytometry "We found that, in agreement with our previous investigation, stimulation of Kv11.3 strongly upregulated both senescent markers in melanoma cells. However, in contrast with the effect of NS1643 in breast cancer cells in which we detected an arrest of the cell cycle in G0/G1 phase, analyses of the cycle progression in A375 melanoma shows that stimulation of Kv11.3 channel significantly increased the population of cells in the G2/M phase compared to untreated cells suggesting that NS1643 determined an arrest of the cell cycle in the G2/M phase of the cell cycle." -- -- -- -- Raf-MEK-ERK -- Western blot "Interestingly, we found that concomitant to the effect of NS1643 on cell cycle proteins, NS1643 strongly inhibited both MEK and ERK activities as indicated by the decreased phosphorylation of these proteins." Human L cellular senescence 26942884 Sen_G_0080 CIP2A 57650 protein coding HCC -- Hepatocellular carcinoma Prevent Flow cytometry//SA-β-gal activity assay//Western blot//Knockdown "The percentage of G0/G1 phase was significantly increased in CIP2A shRNA transfected cells, compared with empty vector group.Our data revealed that the SA-β-gal positive cells were significantly increased in CIP2A shRNA transfected HCC cells." -- -- -- -- -- -- -- -- Human HL cellular senescence 29175329 Sen_G_0081 SFRP1 6422 protein coding IMR-90 -- Aging Accelerate BrdU assay//Immunostaining//SA-β-gal activity assay//Cell morphological analysis//SAHF//RT-PCR "We then tested whether SFRP1 upregulation induces senescence. Lentiviral SFRP1 expression in IMR-9 cells caused striking proliferation arrest as determined by the lack of BrdU incorporation and Ki-67 staining , which was accompanied by senescence phenotypes including flat and enlarged morphology , senescence-associated β gal activity , senescence-associated heterochromatic foci, and induction of genes representing senescence-associated secretory phenotype." -- -- -- -- Wnt-β-catenin//Rb//p53 Downregulation//Activation//-- Western blot//Luciferase reporter assay//Knockdown//SA-β-gal activity assay "Lentiviral SFRP1 expression in IMR-90 cells resulted in reduced soluble βcatenin. Interestingly, etoposide treatment of IMR-90 cells, which stimulates SFRP1 secretion, also reduced soluble β-catenin levels.abolished etoposide-induced reduction of soluble β-catenin levels, suggesting that SFRP1 mediates the downregulation of Wnt signaling upon etoposide treatment. Suppression of Wnt signaling by SFRP1 and etoposide treatment was also verified by Wnt-responsive reporter assays: Wnt3 efficiently activated Super TOPFLASH reporter in IMR-90 cells, which was almost completely repressed by co-expression of SFRP1.Lentiviral SFRP1 expression in IMR-90 cells resulted in dephosphorylation of Rb, but the expression of p53 and a p53 transcriptional target, p21, remained unchanged, suggesting that SFRP1 activates the Rb pathway, but not the p53 pathway. Rb and p53 were efficiently silenced by corresponding shRNAs and upon SFRP1 expression, we observed significantly attenuated senescence induction in cells with Rb or p53 knock-down. This suggests that in addition to Rb, p53 is required for SFRP1-induced senescence even though SFRP1 does not activate the p53 pathway." Human L cellular senescence 22927647 Sen_G_0082 EZH2 2146 protein coding HCT116 -- Colorectal cancer Prevent SA-β-gal activity assay "Consistently, LDMcaused senescence was reduced by overexpressed EZH2 level." p21 -- Western blot//SA-β-gal activity assay "We then checked whether EZH2 knockdown may affect p21 expression, as well as senescent phenotype. Three siRNAs caused apparent reduction of EZH2 expression in both cells, and all the siRNAs significantly increased p21 expression in SW620 cells, whereas two of them (2# and 3#) induced marked p21 expression in HCT116 cells. Although si-EZH2 (1#) failed to increase p21 expression in HCT116 cells for unknown reason, this result confirms that EZH2 negatively regulated p21 expression. Furthermore, EZH2 knockdown with siRNA (3#) caused senescent phenotype, indicating that EZH2 may mediate LDM-induced senescence as well." -- -- -- -- Human HL cellular senescence 27882937 Sen_G_0083 RRM2 6241 protein coding SKOV3 EOC -- Epithelial ovarian cancer Prevent SA-β-gal activity assay "Indeed,knockdown of RRM2 in SKOV3 EOC cells significantly increased SA-βgal activity compared with control cells." -- -- -- -- -- -- -- -- Human HL cellular senescence 24200970 Sen_G_0084 SUV39H1 6839 protein coding WI-38 -- Aging Prevent SA-β-gal activity assay//Cell viability assay//Western blot "In order to test this, we treated cells at different PD levels with chaetocin, a specific inhibitor of SUV39H1 .First, we tested the cytotoxicity of increasing concentrations of chaetocin in the cells for all three PD levels, and found that cell cultures of all senescence states maintained a high level of viability when treated with 5 or 1 nM chaetocin and decreased rapidly at higher concentrations . Interestingly, the PD 54 cells, which express lower levels of SUV39H1, were more tolerant to higher concentrations of chaetocin, as seen by their viability at 48 h and 96 h following chaetocin treatment. In addition to a decrease in viability, increasing concentrations of chaetocin induced greater senescence in PD 47 cells." -- -- -- -- -- -- -- -- Human HL cellular senescence 25063769 Sen_G_0085 CCN1 3491 protein coding MyoFibroblast -- Liver disease Accelerate SA-β-gal activity assay "Numerous senescent cells were found surrounding the central veins in CCl4-treated Ccn1flox/flox mice as judged by the expression of senescence-associated β-galactosidase (SA-β-Gal) as a marker, but their numbers were reduced by ＞6 % in Ccn1△Hep mice ,indicating that CCN1 is critical for senescence." α6β1 Binding SA-β-gal activity assay//Cell counting//qRT-PCR "First, the CCN1-DM mutant protein, which is disrupted in its α6β1 binding sites, was defective for the senescence-inducing activity since it did not suppress cell proliferation or induce SA-β- Gal expression. Second, coincubation of CCN1 with a function-blocking monoclonal antibody against integrin α6 (GoH3), but not control IgG, inhibited CCN1-induced senescence.Finally, an α6β1-binding peptide that competes with ligand binding to α6β1 abrogated CCN1-induced senescence, whereas a mutated T1 peptide that does not bind α6β1 had no effect. Moreover, CCN1 also induced cellular senescence in an α6β1-dependent manner in activated human HSCs. We also isolated PFs, which expressed elastin but not desmin(data not shown), and found that CCN1 triggered cellular senescence in activated PFs as it suppressed cell proliferation and induced the expression of SA-β-Gal and SASP." RAC1-NOX1-ROS -- RT-PCR//qRT-PCR//SA-β-gal activity assay//DHC//Knockdown "CCN1 treatment specifically elevated Nox1 and Rac1 expression, but not expression of other Nox or Rac isoforms or of the Nox2 cofactor p47phox. Knockdown of either Nox1 or Rac1 by specific siRNAs inhibited CCN1-induced ROS and senescence as judged by cell proliferation and SA-β-Gal staining. Knockdown of Nox4, which is also expressed in fibroblasts, had no effect. Likewise, in activated PFs CCN1 induced ROS accumulation and senescence, which also required NOX-depen- dent ROS generation. These results show that CCN1 binds to integrin α6β1and induces cellular senescence through the RAC1-NOX1-ROS axis, leading to the expression of antifibrosis genes associated with the SASP to inhibit fibrosis." Human L cellular senescence 23508104 Sen_G_0086 MAGEA2 4101 protein coding WI-38 -- Tumor Prevent SA-β-gal activity assay "Analysis of senescenceassociated β-galactosidase activity (SA-β-Gal), a well-known marker of senescence,demonstrated that MageA2 expression significantly reduced SA-β-Gal levels in PMLIV-expressing cells." PMLIV -- Western blot "Expression of MageA2 together with PMLIV/p300 strongly decreased PMLIV acetylation. Importantly, siRNA-mediated downregulation of MageA2 in U2OS cells stably expressing PMLIV resulted in increased levels of sumoylated PMLIV, demonstrating the involvement of endogenous MageA2 in the regulation of PMLIV sumoylation." PML-p53 Upregulation Cell morphological analysis//qRT-PCR//SA-β-gal activity assay//BrdU assay//Western blot "To this end, normal human fibroblasts were co-transduced using retroviral vectors expressing PMLIV with an empty vector or in combination with MageA2 or alternatively MageA4. After 10 days under selective culture conditions, PMLIV transduced cells showed all the features of the senescence process, because they ceased to proliferate at sub-confluent densities and became flat and enlarged. Importantly, cells co-expressing PMLIV and MageA2 did not show major morphological changes and behaved similarly to control cells. Colocalization between MageA2 and PMLIV was observed in these cells (data not shown). Analysis of senescenceassociated β-galactosidase activity (SA-β-Gal), a well-known marker of senescence, demonstrated that MageA2 expression significantly reduced SA-β-Gal levels in PMLIV-expressing cells. Moreover, cells expressing MageA2 and PMLIV showed higher levels of BrdU incorporation with respect to those expressing only PMLIV. Cells overexpressing MageA2 alone behaved indistinguishably from control or MageA4 transduced cells. Importantly, MageA2 expression correlated with reduced p21 protein levels, probably due to an effect of MageA2 on efficient p53 activation.Moreover, MageA2 expression affected p53 activity in this cellular system, as assessed by quantification of p53 target genes, thus confirming that MageA2 downregulates the p53-dependent response." Human L cellular senescence 22117195 Sen_G_0087 FOXO3 2309 protein coding HDF -- Aging Prevent SA-β-gal activity assay//Cell morphological analysis//Western blot//Knockdown "Senescence Phenotypes in FOXO3a-siRNA Cells Senescent cells are resistant to mitogen-induced proliferation , express senescence-associated β-gal, and have a characteri stically enlarged, flattened morphology. We measured cell growth and senescence-associated β-gal activity in FOXO3a-siRNA cells. FOXO3a-siRNA cells had an enlarged, flattened cell morphology, like old cells. The percentage of blue cells (indicating senescence-associated β-gal activity) was higher in FOXO3a-siRNA cells than in vectortransfected cells.As expected, the levels of p53 and p21 proteins were up-regulated in FOXO3a-siRNA cells as well as in old cells ." -- -- -- -- -- -- -- -- Human L cellular senescence 15741276 Sen_G_0088 AGT 183 protein coding HUVEC -- Atherosclerosis Accelerate SA-β-gal activity assay//Flow cytometry//Transmission electron microscopy//Cell morphological analysis "SA-β-gal staining was significantly increased in Ang II-stimulated cells in contrast to the control cells which indicates that Ang II promoted cellular senescence in HUVEC.We found that the cell cycle was at G0-G1, the S phase and G2/M phase tended to disappear in Ang II-induced cells compared with the control cells. Transmission electron microscopy was used to evaluate the ultra-microstructure of HUVEC. Senescent cells appeared flattened and enlarged. Chromatin was condensed at the nuclear margin, invagination of the nuclear membrane and vacuolization of the cytoplasm identified aging cells .But the control cell appeared round and smooth in shape and possessed even chromatin." Bcl-2//ERK Downregulation//Activation Immunocytochemical staining//RT-PCR//Western blot "The decrease in Bcl-2 content during aging.Exposure of cells to 10-6mol L 1 Ang II resulted in activation of ERK as manifested by an increase in ERK phosphorylation. The increased phosphorylation of ERK peaked at 12 h after exposure to Ang II and lasted for 36 h. However, the activity of p-ERK declined to the basal level at 48 h after Ang II treatment." -- -- -- -- Human L cellular senescence 18383564 Sen_G_0089 TGFB1 7040 protein coding HCEC -- Aging Accelerate SA-β-gal activity assay//Flow cytometry//qPCR//Western blot "Cell cycle analysis by flow cytometer showed that the HCECs accumulated at G1 phase with a concomitant depletion of S phase cells after TGF-β1 exposure, suggesting that cell cycle arrest during HCECs senescence induced by TGF-β1 occurred at G1 phase, while H2O2 induced an obvious G2/M phase arrest. In association with the G1 arrest, we also found TGF-β1 increased the percentage of SA-β-gal staining cells and concomitantly increased the expression of p16 and p21, as analyzed by real-time PCR or western blot. In order to further confirm the effect of TGF-β on cellular senescence, we interrupted the TGF-β signaling pathway using a specific inhibitor, LY364947. When treated with LY364947, the percentage of SA-β-gal staining cells was significantly decreased, and the levels of p21 and p16 were also downregulated." -- -- -- -- NF-κB Activation Western blot "Compared with pre-senescent cells, TGF-β1 treatment led to significant reduction in IκBα protein levels. IκBα is a canonical and specific inhibitor of NF-κB, and its reduction suggested elevated NF-κB activity in TGFβ-induced senescent cells; our subsequent results confirmed this. IκBα loss is the key step that immediately releases cytoplasmic NF-κB for nuclear translocation and the subsequent phosphorylation of p65, a subunit of NF-κB. Therefore, we measured the phosphorylation of p65 in HCECs treated with or without TGF-β. Much more phosphorylation of p65was found in TGF-β-treated HCECs than in untreated cells." Human L cellular senescence 27713146 Sen_G_0090 CSNK2A1 1457 protein coding HCT116 -- Aging Prevent SA-β-gal activity assay "To detect senescence, p53-/- and wild-type HCT116 cells were treated with CKII inhibitors, apigenin and DRB, and then stained for SA-β-gal activity. In the p53 positive HCT115 cells, there was a significant dosedependent increase in SA-β-gal activity in response to apigenin and DRB." p52//Rb --//-- SA-β-gal activity assay//Western blot "However, p53 negative HCT116 cells showed only slight signs of senescence demonstrating that p53 is required for the induction of senescence through CKII inhibition.The level of hyperphosphorylated RB protein decreased while hypophosphorylated form increased in wildtype HCT115 cells treated with CKII inhibitor." p53-p21 Activation SA-β-gal activity assay//Western blot//RT-PCR "When these transfectants were stained for SA-β-gal activity, p53 negative HCT116 cells exhibited an apparently lower rate of SA-β-gal activity compared to p53 positive HCT116 cells. In p21Cip1/WAF1 positive HCT116 cells, there was a significant increase in SA-β-gal activity in response to apigenin and DRB. However, p21Cip1/WAF1 negative HCT116 cells showed only slight signs of senescence demonstrating that p21Cip1/WAF1 is required for the induction of senescence via CKII inhibition. Cells treated with apigenin showed an increase both in the p21Cip1/WAF1 protein and mRNA levels, suggesting that CKII inhibition up-regulates p21Cip1/WAF1 expression at the transcriptional level ." Human L cellular senescence 19855935 Sen_G_0091 BAG2 836330 protein coding TRE293 -- Aging Accelerate Western blot//BrdU assay//SA-β-gal activity assay//SAHF "To assess the functional role of BAG2 in senescence, we generated a TRE293 cell line with inducible BAG2 (TRE293-BAG2) in which the BAG2 expression is triggered by the tetracycline regulatory (Tet-on) system. Like other senescent cells, the TRE293-BAG2 cells exhibited cell cycle arrest and slow proliferation and a significant increase in SA-β-Gal staining. In a senescence cell, it is basically accepted that the abundant enhancement of senescence-associated heterochromatic foci (SAHF) with heterochromatin proteins such as HP1 represses expression of proliferation-promoting genes. The senescence induced by BAG2 was further confirmed by examining changes in the chromatin structure." -- -- -- -- p21 Activation Western blot//Knockdown//SA-β-gal activity assay "We monitored the levels of typical biomarkers of senescence in TRE293-BAG2 cells, (p21CIP1, p16INK4a, or p27KIP1). Western blotting revealed no significant change in the levels of p16INK4a or p27KIP1. However, there was a strong accumulation of p21CIP1, suggesting that the senescence induced by BAG2 was likely via the p21CIP1 pathway, which was also observed in H520-BAG2 cells. This interpretation was further supported by BAG2 knock-down experiments.The level of p21CIP1 protein was reduced in the TRE293 cells treated with BAG2 small interfering RNA (siRNA). To determine if the senescence induced by BAG2 was mediated only through the p21CIP1 pathway, we transfected p21CIP1 shRNA into TRE293- BAG2 cells. We found that senescence was almost fully restored when p21CIP1 was down-regulated." Human HL cellular senescence 22146591 Sen_G_0092 GRK4 2868 protein coding HEK293 -- Aging Accelerate SA-β-gal activity assay//Immunostaining "The Ki-67 protein were clearly detected within the cell nuclei of GRK4(-) cells and CNE2 cells, whereas it was barely found in GRK4(+) cells. We next assessed the SA-β-gal activity in the GRK4(+) and GRK4(-) cells by a SA-β-gal staining. The stained cells and total cells were counted under an inverted phase microscope and the percentage of cells staining positive was calculated.High stain positive of 99±0.20% in GRK4(+) cells for SA-β-gal was detected , while this was markedly low to only1±0.15% in GRK4(-) cells." -- -- -- -- -- -- -- -- Human L cellular senescence 28912086 Sen_G_0093 TAK1 39659 protein coding IMR-90 -- Aging Prevent SA-β-gal activity assay The number of cells expressing SA-β-gal was dramatically increased after treatment with the TAK1 inhibitor relative to the number in the control cells. p53 -- Western blot "p53 expression in RPE cells under oxidative stress was strongly affected by pretreatment of the cells with the TAK1 inhibitor, seen by the inhibition of p38 phosphorylation . In control cells (without such pretreatment) the expression of p53 gradually increased, reaching a peak after 60 minutes, whereas in the pretreated cells, p53 expression peaked after 10 minutes and then declined.Over a longer period, TAK1 inhibition reduced p53 expression to a level slightly higher than in control cells." -- -- -- -- Human L cellular senescence 25118260 Sen_G_0094 BTG3 492479 protein coding HCT116 -- Aging Prevent SA-β-gal activity assay//Flow cytometry//BrdU assay//Western blot//Cell morphological analysis//Knockdown "In contrast to what was observed previously in HCT116 cells,loss of BTG3 expression in IMR90 cells resulted in blunted cell proliferation, as demonstrated by a significant decrease of cells in the S phase, a moderate increase in the G1 population, a marked reduction in BrdU incorporation and RB phosphorylation. The slowed proliferation remained for at least for 2 weeks , indicating that this is not a transient growth arrest. Additionally, BTG3-knockdown cells displayed the enlarged, flattened morphology that is typical of senescent cells . The phenotype was confirmed by staining the cells for senescenceassociated β-galactosidase (SA-β-Gal) activity: we observed an increase in SA-β-Gal staining of at least sixfold 48 h after transfection with the BTG3-2 siRNA , which was further enhanced 1 week after transfection." JMJD3 -- RT-PCR//Knockdown "Using RT PCR, we found that the expression of JMJD3 was increased in BTG3-knockdown cells." ERK-AP1 -- RT-PCR//SA-β-gal activity assay//Knockdown "To confirm the involvement of the ERK signaling pathway, we treated BTG3-knockdown IMR90 cells with the ERK-specific inhibitor PD98059. As a result, a significant reduction in senescence was observed when ERK was inhibited. In contrast, two other unrelated drugs, SB202190 (p38 inhibitor) and SB202474 (negative control), had no apparent effect, indicating the specificity of the treatment." Human HL cellular senescence 22020331 Sen_G_0095 RRN3 54700 protein coding "MCF-7,MEF" -- Aging Accelerate SA-β-gal activity assay//qRT-PCR//Western blot//Immunostaining "We found that RPL11 depletion suppressed the accumulation of p53, p21, and p16, and the number of SA-β-gal-positive cells induced by TIF-IA overexpression . We depleted RPL11 in the presence of small interfering RNAs (siRNAs) specific for DKC1, RRP5, and RRS1, any of which induced p53 accumulation, p21 induction, p16 induction, and cellular senescence.We found that RPL11 depletion abrogated these events." -- -- -- -- p53-p21 Activation SA-β-gal activity assay "As with the case of TIF-IA overexpression, the increased number of SA-β-positive cells by the depletion of the rRNA-processing factors was counteracted by p53 or p21 depletion ." Human L cellular senescence 25732822 Sen_G_0096 PON1 5444 protein coding HCT116 -- Aging Prevent SA-β-gal activity assay//Knockdown "To evaluate the influence of PON1 knockdown on ageing of HDMECs, we measured cellular senescence by SA-β-gal staining. Cell senescent morphology, such as an enlarged and flattened shape with an increased diameter, was observed in aged HDMECs at passage 2 and in PON1 siRNA #2-treated HDMECs at 48 h post-transfection. The number of cells positive for SA-β-gal staining increased significantly compared to early passage HDMECs at passage 8 and in scrambled siRNA-treated HDMECs." p16 -- Western blot "In Western blot, the expression of p16 in PON1 knockdown HDMECs was observed. At 48 h post-transfection, the protein expression of p16 in PON1 siRNA-treated HDMECs was significantly higher than the expression level in scrambled siRNA-treated HDMECs." -- -- -- -- Human L cellular senescence 22897574 Sen_G_0097 SMARCA4 6597 protein coding SW48 -- Colorectal cancer Prevent Western blot//qRT-PCR//SA-β-gal activity assay//CCK-8 assay//Flow cytometry//DAPI staining "Data showed that the percentages of SA-β-galpositive cells in SW48 sh-BRG1 cells significantly increased compared with sh-Con cells in both DOX treatment and nontreatment conditions .The results showed that the growth rates significantly decreased in BRG1 KD groups, suggesting that BRG1 KD inhibited cell proliferation. Cell cycle analysis by flow cytometry detection was performed, and the results revealed that BRG1 KD led to cell cycle arrest at G2-M phase. Furthermore, we examined SAHF by staining 4,6-diamidino-2-phenylindole (DAPI), which showed distinct SAHF formation in BRG1 KD cells as observed by laser confocal fluorescence microscopy scanning." -- -- -- -- SIRT1-p53-p21 -- Western blot//qRT-PCR//IHC//SA-β-gal activity assay//Co-IP//Immunofluorescence "Our data showed that the increase of p21 induced by BRG1 KD was abrogated by the treatment of p53 siRNA. Moreover, SA-β-gal-positive cells stopped increasing after the KD of BRG1 with p53 siRNA in both DOX treatment and non-treatment conditions.The regulation of BRG1 in the p53/p21 pathway was further confirmed by using another shRNA against BRG1. These results suggested that KD of BRG1 promotes senescence by activating the p53 pathway in CRC cells." Human L cellular senescence 28182012 Sen_G_0098 PPARD 5467 protein coding Primary keratinocyte -- Skin cancer Accelerate SA-β-gal activity assay//BrdU assay "A higher percentage of BrdU labeling and lower percentage of β-gal-positive cells was noted in HRAS-expressing PPARβ/δ-null compared with wild-type cells. Surprisingly, ligand activation of PPARβ/δ decreased both the percentage of β-gal- and BrdU-positive cells in HRAS-expressing wild-type but not in PPARβ/δ-null cells." RASGRP1//ILK --//Downregulation Western blot//qPCR//CHIP "Expression of the negative RAS regulator RASGAP120 was increased and the positive RAS regulator RASGRP1 was decreased in response to HRAS activation, respectively .Interestingly, expression of RASGAP120 was significantly higher, whereas expression of RASGRP1 was lower in HRAS expressing PPARβ/δ-null cells compared with wild-type cells. In response to HRAS activation, expression of mRNA encoding the negative RAS regulator Rasa4 was increased in cells from both genotypes, but relatively higher expression of Rasa4 mRNA was also observed in HRAS-expressing PPARβ/δ-null cells compared with wild-type cells (data not shown).Consistent with past studies,32 34 expression of ILK and PDPK1 was higher in control and HRAS-expressing PPARβ/δ-null cells. In silico examination of the Ilk gene revealed two potential PPREs in the second intron .Interestingly, mutating the PPARβ/δ binding half-site in a luciferase reporter construct containing the upstream PPRE abolished the repression of ILK by PPARβ/δ." PI3K-Akt//MEK-ERK Downregulation//Upregulation Western blot//SA-β-gal activity assay//Immunofluorescence//Histochemical staining//Cumulative frequency of mean intensity of cells "The higher levels of p-MEK and p-ERK and lower level of p-AKT in wild-type cells correlated with higher markers of senescence and this was not found in HRAS-expressing PPARβ/δ-null cells . Inhibition of MEK1 with PD98059 delayed HRAS-induced senescence in both wild-type and PPARβ/δ-null cells .Higher expression of ILK, p-AKT (S473, T308) was also found in skin tumors from PPARβ/δ-null mice compared with wild-type mice." Human HL cellular senescence 24213576 Sen_G_0099 HDAC1 3065 protein coding UCB-MSC -- Aging Prevent SA-β-gal activity assay "Consistently with our previous report, the inhibition of HMGA2 as well as HDAC1 and HDAC2 led to the senescence of hUCB-MSCs as shown by SA β-gal staining, in addition to significantly increased expression of p21CIP1/WAF1 and p16INK4A." -- -- RT-PCR//qRT-PCR//SA-β-gal activity assay "Consistently with our previous report, the inhibition of HMGA2 as well as HDAC1 and HDAC2 led to the senescence of hUCB-MSCs as shown by SA β-gal staining, in addition to significantly increased expression of p21CIP1/WAF1 and p16INK4A." -- -- -- -- Human HL cellular senescence 20652617 Sen_G_0100 HDAC2 3066 protein coding UCB-MSC -- Aging Prevent SA-β-gal activity assay "Consistently with our previous report , the inhibition of HMGA2 as well as HDAC1 and HDAC2 led to the senescence of hUCB-MSCs as shown by SA β-gal staining, in addition to significantly increased expression of p21CIP1/WAF1 and p16INK4A." -- -- RT-PCR//qRT-PCR//SA-β-gal activity assay "Consistently with our previous report [21], the inhibition of HMGA2 as well as HDAC1 and HDAC2 led to the senescence of hUCB-MSCs as shown by SA β-gal staining, in addition to significantly increased expression of p21CIP1/WAF1 and p16INK4A." -- -- -- -- Human HL cellular senescence 20652617 Sen_G_0101 BHLHE40 8553 protein coding MCF-7 -- Aging Accelerate SA-β-gal activity assay//Western blot Microscopic analysis showed that the number of SA-β-galactosidase-positive colonies was increased in DEC1-expressing cells compared with that in control and mutant DEC1-expressing cells. p53 -- Northern blot "To confirm this, Northern blot analysis was performed. We showed that DEC1 was induced by p53 but not mutant p53(R249S) in H1299 cells." -- -- -- -- Human HL cellular senescence 18025081 Sen_G_0102 SLC13A3 64849 protein coding "MRC-5,WI-38" -- Aging Accelerate SA-β-gal activity assay//Immunostaining "In the present study, the positive rate of SA-β-gal staining has reached 95% in the NaDC3-infected MRC-5 cells at PD 37, which was significantly increased than those in the uninfected and control vector infected cells. When examining cellular morphology of the infected cells, we observed significant senescent morphological changes in the NaDC3-infected cells. The results by DAPI staining showed that NaDC3 could also induce similar premature cellular senescence in the WI-38 cells." NADH Upregulation NAD+/NADH Quantitation kit//Confocal microscope NaDC3 overexpression markedly increased intracellular NADH levels compared with the two controls. -- -- -- -- Human L cellular senescence 25384549 Sen_G_0103 DIRAS3 9077 protein coding ASC -- Aging Prevent Knockdown//SA-β-gal activity assay//Cell morphological analysis "DIRAS3 KD cells displayed a cytoplasm with thin and long processes and a significant number of these cells became flat, resembling the morphological features of the premature senescence phenotype induced by oncogenic assault in primary human fibroblasts . As demonstrated by cytochemical staining and FACS analyses using a fluorogenic substrate C12FDG,DIRAS3 KD led to a significant increase in the number of senescenceassociated β-galactosidase (SA-β-GAL) positive ASCs and to a significant increase in cell size ." -- -- -- -- Akt-mTOR Downregulation Western blot//qRT-PCR//Knockdown To investigate the effect of DIRAS3 knock-down (KD) on Akt-mTOR signaling in ASCs we employed lentivirus mediated DIRAS3 specific shRNA . DIRAS3 KD leads to increased activity of Akt-mTOR signaling in ASCs. Human L cellular senescence 28316325 Sen_G_0104 EZH2 2146 protein coding SKOV3/DDP -- Ovaria cancer Prevent SA-β-gal activity assay//Western blot "The inhibition of EZH2 expression significantly downregulated H3K27me3 expression and upregulated p14, p16, p53, and pR protein expression. This suggested that EZH2 inhibition led to the activation of the INK/ARF/Rb pathway and, thereby, induced cellular senescence. Furthermore, microscopic analyses revealed the following morphological changes in the SKOV3/DDP cells in the pSUPER-EZH2 group: increased cell volume, flat granules, and β-galactosidase staining (blue), which are characteristics of cellular senescence. These changes were not observed in the blank control group." -- -- -- -- INK-ARF-Rb -- Western blot "The inhibition of EZH2 expression significantly downregulated H3K27me3 expression and upregulated p14, p16, p53, and pR protein expression. This suggested that EZH2 inhibition led to the activation of the INK/ARF/Rb pathway and, thereby, induced cellular senescence." Human HL cellular senescence 27610467 Sen_G_0105 UBE3A 7337 protein coding Human Burkitt Lymphoma cell line derived cell line -- B-cell lymphoma Prevent SA-β-gal activity assay//Flow cytometry "Down-regulation of E6AP increased the proportion of cells in G1 with a corresponding decrease of cycling cells , resulting in a reduction in cell proliferation with no impact on cell viability. This effect coincided with a significant number of cells showing hallmarks of senescence ." -- -- -- -- PML Upregulation Western blot//Immunofluorescence "Strikingly, E6AP knockdown resulted in restoration of PML expression and formation of PML-NBs in these cells." Human L cellular senescence 22689861 Sen_G_0106 DNMT1 1786 protein coding UCB-MSC -- Aging Prevent SA-β-gal activity assay//MTT assay "DNMT inhibition by 5-AzaC treatment induced cellular senescence, as shown by SA β-gal staining , and decreased the cellular proliferation rate in a dose-dependent manner, as shown by an MTT assay." p16//p21/WAF1//EZH2//BMI1//miR200c//miR-214 --//--//--//--//--//-- SA-β-gal activity assay//qPCR//Western blot "SA β-galactosidase activity and expression levels of p16INK4A and p21CIP1/WAF1 were increased at day 3 of 5-AzaC treatment, and prominent changes were observed after 5 days of treatment with 5- AzaC.After inhibition of DNMT by 5-AzaC treatment, we observed decreased EZH2 and BMI1 expression levels. Because the significant decrease of EZH2 and BMI1 occurs after 3 days of treatment with 5-AzaC, we investigated miRNA expression levels at 1, 3 and 7 days after treatment with 5-AzaC and found that both mature and precursor miRNAs were increased at the time points indicated. After overexpression of miR-200c and miR-214, MSCs underwent cellular senescence, as shown by SA β-gal staining, and BMI1 and EZH2, the respective targets of miR200c and miR-214 were decreased, as shown by real-time qPCR. In addition, inhibition of miR-214 using antisense oligonucleotide transfection increased EZH2 expression. Although inhibition of miR-200c did not yield consistent results, overexpression of both miRNAs decreased their respective target (BMI1 and EZH2) at the mRNA level, suggesting that overexpressed miRNA during cellular senescence regulates the expression levels of BMI1 and EZH2." -- -- -- -- Human HL cellular senescence 21572997 Sen_G_0107 SIRT1 23411 protein coding 2BS -- Aging Prevent SA-β-gal activity assay//Immunostaining 2BS with enforced expression of wild type SIRT1 displayed lower frequency of SA-β-gal staining than cells transfected with mock vector and H363YSIRT1 constructs. We observed that 2BS cells expressing SIRT1 did not develop pronounced nucleoli or DNA foci. -- -- -- -- p16-Rb//ERK-S6K1 --//Activation Western blot "Compared with mock vector and H363YSIRT1 transfected 2BS cells, p16INK4A displayed a two-fold decrease in SIRT1-transfected cells, accordingly phospho-Rb (Ser795) was increased for more than two times whereas the levels of b-actin and Rb remained unchanged in different batches of transfected cells.The levels of SIRT1 protein were declined in late passage of 2BS (63PDs), and the phosphorylations of ERK and S6K1, but not that of AKT, were also decreased in senescent cells." Human L cellular senescence 18320031 Sen_G_0108 TBX2 6909 protein coding WI-38 -- Aging Prevent SA-β-gal activity assay//Immunostaining "Additionally, TBX2/PML-IV-expressing cells stained positive for Ki67 and negative for SA-β-Gal when compared with DON/PML-IV cells." PML Binding qRT-PCR//Western blot We detected a robust increase both in TBX2 transcript (~4-fold) and in protein levels (B3-fold) in PML-/- MEFs when compared with PML+/+ MEFs as determined by qRT-PCR (left panel) and western blot analysis (right panel). -- -- -- -- Human HL cellular senescence 22002537 Sen_G_0109 NUAK1 9891 protein coding WI-38 -- Aging Accelerate Crystal violet assay//SA-β-gal activity assay "NUAK1 expression was found to block cell growth and induce a characteristic senescent morphology. This growth arrest was due to premature senescence induction, as evidenced by the increased proportion of SA-β-Gal-positive and SAHF-positive cells among the NUAK1-overexpressing cells, as compared with control cells ." LKB1//LATS1 Activation//Downregulation Crystal violet staining//IP "The growth-inhibitory effect of NUAK1 was largely reverted when LKB1 activity was inhibited by either expression of a dominantnegative form or by its knockdown .NUAK1 and LA TS1by performing co-immunoprecipitation assays on extracts from cells co-expressing NUAK1 and LATS1. Interestingly, NUAK1 was detected in the LATS1 immunoprecipitate. We next repeated the immunoprecipitation experiment with endogenous proteins. Once again, NUAK1 protein was found in the LATS1 immunoprecipitate." -- -- -- -- Human HL cellular senescence 19927127 Sen_G_0110 LATS1 9113 protein coding WI-38 -- Aging Prevent Crystal violet assay//Hoechst staining//SA-β-gal activity assay "A growth arrest was observed when compared with control infected cells, as judged using colony formation assays.Similar to constitutive NUAK1 expression, the LATS1 DN expression induced gross aneuploidies and the appearance of SA-β-Gal activity." -- -- -- -- -- -- -- -- Human HL cellular senescence 19927127 Sen_G_0111 GDF15 9518 protein coding TBE -- Aging Accelerate SA-β-gal activity assay "Disruption of GDF15 markedly reduced CSEinduced cellular senescence, represented by less induced SA β-gal activity, less inhibition of cell proliferation, and less HMGB1 release." -- -- -- -- ALK1-Smad1 Activation Western blot "GDF15 protein time-dependently induced phosphorylation of Smad1/5/8 in primary human airway epithelial cells. Moreover, a direct binding assay of GDF15 and ALK1 protein was performed in vitro to confirm the direct interaction between GDF15 and ALK1. We found that GDF15 bound to the ALK1 receptor in a dose-dependent manner.In our pilot study, CSE significantly increased GDF15, HMGB1, p21 and Smad1/5/8 phosphorylation at day 1 in hTBE cells." Human HL cellular senescence 27093475 Sen_G_0112 AKTIP 64400 protein coding Fibroblast -- Aging Prevent SA-β-gal activity assay//Knockdown Cell labelling with the empirical senescence marker SA-βgal revealed that early passage HPFs at 11 and 13 days after shAKTIP infection exhibit a significant increase in senescent cells compared with controls. -- -- -- -- -- -- -- -- Human L cellular senescence 27512140 Sen_G_0113 PPM1B 5495 protein coding IMR-90 -- Aging Prevent SA-β-gal activity assay//Western blot//Cell morphological analysis "In contrast to the basal level of staining observed in the control cells, the PPM1B depleted cell lines developed a characteristic senescence-associated β-GAL staining pattern with a 10-fold increase in the percentage of stained cells. Moreover PPM1B depleted cells displayed a flattened and irregular cell shape with a bigger cell volume which is typical of senescent cell morphology . In addition, the gene expression levels of four senescence markers were examined by RT-qPCR in these cells . The transcript abundance of p21, plasminogen activator inhibitor 1 (PAI), SA-βgalactosidase (β-GAL) and p16 all showed a two to threefold increase in gene expression upon PPM1B depletion compared to those infected with control virus. The increase in transcript abundance is consistent with the immunoblot analysis against of PAI-1, p21 and p16, suggesting that the upregulation of senescence markers occurs at both the transcript and protein levels." -- -- -- -- -- -- -- -- Human HL cellular senescence 24674756 Sen_G_0114 RPS14 6208 protein coding IMR-90 -- Aging Accelerate SA-β-gal activity assay//Western blot "Next, we characterized the effects of directly decreasing the ribosome biogenesis factors RSL1D1, NS, DDX21 or EBP2 using shRNAs. Depletion of these factors induced a proliferation arrest, a decrease in the proliferation markers KI67 and CENPA, and an increase in senescence-associated β-galactosidase staining (SA-β-gal)." CDK4//Cyclin D1 --//-- Pull-down assay GST pull down assays with purified proteins revealed that RPS14 binds to either CDK4 or cyclinD1 alone or to the complex of CDK4 with cyclinD1. Rb -- Pull-down assay//SA-β-gal activity assay//Western blot//Knockdown "The E7 mutant Δ79 83, which binds and inhibits Rb, rescued cells from senescence after knockdown of RSL1D1, while mutants E7Δ6 10 and E7Δ21 24 that do not bind Rb were not efficient at inhibiting senescence after knockdown of RSL1D1. Furthermore, the kinases CDK4 and CDK6, which are able to phosphorylate and inactivate Rb29, were also capable of preventing senescence after knockdown of RSL1D1. Expression of intact CDK4, but not of a catalytically inActivate mutant (CDK4(K35M)), efficiently bypassed senescence, preventing the downregulation of Ki67 expression, as well as rescuing Rb phosphorylation and the expression of E2F targets such as CENPA and MCM6 after knockdown of RSL1D1 ." Human HL cellular senescence 29941930 Sen_G_0115 ERBB2 2064 protein coding -- Prostate Prostate cancer Prevent Ki67 staining//BrdU assay//SA-β-gal activity assay "The PB-Cre4: Ptenfl/fl Her2KI tumors had significantly higher levels of proliferation than the PBCre4: Ptenfl/fl tumors, as assessed by Ki67 and BrdU . Furthermore, there was a marked down-regulation of the growth arrest and senescence markers p16 and p21 in the PB-Cre4: Ptenfl/fl Her2KI compared with the PB-Cre4: Ptenfl/fl mice. Moreover, there was a robust down-regulation of senescence-associated β-galactosidase signals in the PBCre4: Ptenfl/fl Her2KI prostates compared with PB-Cre4: Ptenfl/fl mice ." -- -- -- -- MEK-ERK//PI3K-AKT Activation//-- Western blot "Tumors from PB-Cre4: Ptenfl/fl Her2KI mice showed elevated pERK1/2 activation (with total ERK1/2 levels unchanged), whereas PB-Cre4: Ptenfl/fl tumors had very low levels of pERK1/2. Heregulin treatment also increased PTEN phosphorylation, which can in turn decrease the ability of PTEN to inhibit PI3K-mediated AKT activation ." Human HL cellular senescence 21930937 Sen_G_0116 SIRT3 23410 protein coding WI-38 -- Aging Prevent SA-β-gal activity assay//DAPI staining//Western blot "Our results indicated that the overexpression of SIRT3 significantly reduced the number of cells positive for SA-β-gal and inhibited SAHF formation compared with empty vector .Meanwhile, the accumulation of p16INK4A was decreased ." -- -- -- -- FOXO1 Binding Confocal microscopy//Western blot "We found that the endogenous SIRT3 was highly expressed in the mitochondria and cytoplasm, FOXO1 was expressed in the cytoplasm and nuclei, and the SIRT3 staining strongly overlapped with that of FOXO1 in the cytoplasm. We could confirm that SIRT3 was binding with FOXO1. SIRT3 overexpression keeps FOXO1 acetylation to a very low level.To verify the direct effect of SIRT3 on FOXO1-targeted gene expression including catalase and MnSOD, we examined the expression of target genes. The results indicated that expression levels were increased in the pSIRT3 group compared with control and vector groups." Human L cellular senescence 23494737 Sen_G_0117 AKT1 207 protein coding Primary leiomyoma cell -- Uterine leiomyomas Prevent SA-β-gal activity assay//qPCR "The percentage of β-galactosidase stained DDHLM and primary leiomyoma cells with MK-2206 treatment were significantly higher than untreated cells.Approximately 32% of cells exhibited SAHF when treated with MK-2206 compared to 8% in control cells. When leiomyoma cells were treated with MK-2206, the senescence associated genes,P16,P21 and P53 were significantly upregulated ." miR-182//miR-200//HMGA2 --//--//-- Immunofluorescence//qPCR "In response to MK-2206 as well as the dual PI3K/mTOR inhibitor, BEZ235, ROS levels increased in primary leiomyoma cells. Similarly,miR-182 and miR-200a/c expression increased in leiomyoma cells treated with MK2206 in a dose-dependent manner.HMGA2 expression was significantly increased in a dose-dependent manner when AKT was inactivated by MK-2206." -- -- -- -- Human L cellular senescence 24476133 Sen_G_0118 CPT1C 126129 protein coding PANC-1 -- Pancreatic cancer Prevent BrdU assay//SA-β-gal activity assay//DAPI staining//qPCR "Upon CPT1C loss, the number of PANC-1 cells Activately replicating DNA decreased,consistent with effective inhibition of colony formation in a cell concentrationdependent manner , indicating that PANC-1 cells lacking expression of CPT1C have reduced proliferation. In addition, strong SA-β-gal activity indicated that CPT1C depletion led to the severe Y. Wang et al. senescence state of PANC-1 cells.As expected, loss of CPT1C caused PANC-1 cells to change shape and increase cytoplasmic granularities. DNA SAHF was specifically enriched in CPT1C depleted PANC-1 cells, while controls were excluded from SAHF. CPT1C-depleted PANC-1 cells also had abnormal nuclear size, when compared to control cells.Furthermore, a significant increase in IL8 mRNA levels confirmed a sharp induction of the SA phenotypic changes observed by the SA-β-gal assay. Finally, the TRF length assay revealed that CPT1C depletion inhibited the elongation of telomeres by 0.8 kb in PANC-1 cells." -- -- -- -- -- -- -- -- Human L cellular senescence 29317762 Sen_G_0119 MIF 4282 protein coding MSC -- Aging Prevent qPCR//Cell morphological analysis "Moreover, when comparing cell morphology between MIF-overexpressing and the control cells, the control hMSCs displayed enlarged and flattened morphology, whereas the MIF-overexpressing hMSCs maintained spindle morphology.In addition, our real-time PCR results showed that the expression levels of p16 and p21 mRNA transcripts were significantly downregulated (P < 0.05) in MIF-overexpressing hMSCs compared to those in the control hMSCs. " -- -- -- -- Akt Activation Western blot "In addition to increased expression of MIF, our western blot results also showed that the levels of total and phosphorylated AKT were increased in MIF-overexpressing hMSCs than those in the control cells." Human HL cellular senescence 24274936 Sen_G_0120 IGFBP3 3486 protein coding "MCF-7,IMR-90" -- Aging Accelerate BrdU assay//SA-β-gal activity assay//Ki67 staining "Lentiviral IGFBP3 expression in MCF7 cells caused striking proliferation arrest as determined by the lack of BrdU incorporation and Ki-67 staining, which was accompanied by the induction of SA β-gal activity. Further, addition of purified recombinant IGFBP3 protein to the culture medium of MCF-7 cells resulted in dosedependent induction of SA β-gal activity, increased G1 and decreased S and G2/M fractions, reduced BrdU incorporation , and reduced mitotic phospho-histone H3 staining . Lentiviral IGFBP3 expression in IMR-90 human primary fibroblasts also resulted in abrogation of Ki-67 staining , induction of SA β-gal activity,and formation of senescence-associated heterochromatic foci." Akt -- SA-β-gal activity assay//Western blot "In accordance with this finding, IGFBP3 suppressed the baseline and IGF-induced levels of phosphorylated AKT, which is a downstream target of IGF signaling. We then assessed the role of AKT in IGFBP3-induced senescence by using constitutively Activate AKT (i.e., myr-AKT). myr-AKT expression in MCF-7 cells resulted in elevation of phosphorylated AKT levels and abrogation of IGFBP3-induced senescence, suggesting that IGFBP3 induces senescence through suppression of AKT activity." p53//Rb --//-- SA-β-gal activity assay//Western blot//Knockdown "Senescence induced by IGFBP3 was abolished by knockdown of p53 or Rb, which suggests the requirement of p53 and Rb tumor suppressor pathways for IGFBP3-induced senescence." Human L cellular senescence 22778398 Sen_G_0121 SERPINE1 5054 protein coding MCF-7 -- Aging Accelerate Luciferase reporter assay//SA-β-gal activity assay//Western blot//Knockdown "PAI-1 knockdown resulted in concomitant decrease in extracellular IGFBP3 levels and suppression of senescence induction. These results suggest that doxorubicin-induced DNA damage enhances the secretion of PAI-1, which in turn results in elevated extracellular IGFBP3 and induction of senescence in MCF-7 cells." t-PA//IGFBP3 --//-- Knockdown//Western blot "The reduction of IGFBP3 levels upon PAI-1 knockdown was alleviated by the addition of anti t-PA antibody to the culture medium, supporting the role for t-PA in mediating IGFBP3 reduction upon PAI-1 knockdown.Recombinant PAI-1 increased extracellular IGFBP3 levels and induced senescence in MCF-7 cells, which was abolished by knockdown of IGFBP3." -- -- -- -- Human L cellular senescence 22778398 Sen_G_0122 AGO2 27161 protein coding WI-38 -- Aging Accelerate SA-β-gal activity assay//EdU assay//DAPI staining "In AGO2-deficient cells replicative lifespan increased by ～3–4population doublings whereas E7-expressing cells showed a lifespan extension of～5–6 population doublings. In line with this finding, the fraction of AGO2- deficient cells incorporating EdU increased and senescence-associated beta-galactosidase (SA-β-Gal) activity decreased transiently when compared with shC control cells.Strikingly, AGO2-overexpressing cells induced an abrupt proliferative arrest with features of senescence as shown by a decrease in the number of cells incorporating EdU and an increase in the percentage of SAHF-positive cells." E2F//Rb1//let-7f Binding//--//-- CHIP//Western blot//RIP "This analysis revealed, that of the top 577 E2F-responsive promoters known at present ,320 (that is 55.5%) were occupied by AGO proteins in senescent cells, as opposed to only 77 (that is 13.3%) in control cells.To test this possibility, we first carried out co-immunoprecipitation experiments between endogenous AGO2, RB1 and HDAC1 in cellular lysates prepared from RASV12-induced senescent fibroblasts.HDAC1 and AGO2 co-immunoprecipitated with each other. Moreover, both HDAC1 and AGO2 efficiently co-immunoprecipitated RB1. Similar results were obtained in Saos-2 cells undergoing RB1- induced senescence. Consistent with the physical interaction between AGO2 and RB1,AGO2 showed partial colocalization with SAHFs at their periphery, a staining pattern that was similar for the heterochromatin marker H3K27me3 and RB1 (ref. 2) and is thus congruent with the peripheral SAHF localization of E2F-target genes.The interaction between either H3K9me2 or AGO2 and let-7f was confirmed by RNA immunoprecipitation combined with small RNA complementary DNA (srcDNA) cloning and sequencing in senescent cells." -- -- -- -- Human HL cellular senescence 22366686 Sen_G_0123 YAP1 10413 protein coding MSC -- Osteoarthritis Prevent SA-β-gal activity assay//Flow cytometry//Western blot "Phenotypic characterizations revealed that the downregulation of YAP in hMSCs also resulted in a similar premature aging phenotype. By contrast, ectopic expression of YAP rescued the premature senescence observed in YAP?/?hMSCs, as evidenced by the reduced number of SA-β-gal–positive cells ,enhanced growth rate and clonal expansion ability , decreased expression of P16 and P21, lower levels of ROS , and slower in vivo decay after engraftment." -- -- -- -- YAP-TEAD//YAP-FOXD1 --//-- SA-β-gal activity assay//Dual-Luciferase reporter assay//Western blot//Clonal expansion assay "Similar to YAP-deficient hMSCs, TEADs KD/KO hMSCs also showed major phenotypes of premature senescence, such as an increased number of SA-β-gal–positive cells, compromised clonale. Moreover, the activity of 8 × GTIIC-Luc, a YAP/TAZ-responsive reporter, decreased in both RS hMSCs and WS hMSCs . Lentiviral overexpression of YAP or FOXD1 effectively attenuated the senescent features of RS hMSCs and WS hMSCs." Human HL cellular senescence 30933975 Sen_G_0124 ING1 3621 protein coding PCa -- Prostate cancer Accelerate SA-β-gal activity assay "By conducting SA-β-Gal staining in the stably transduced cells, ING1b was shown to induce cellular senescence in PCa cells . Further SA-β-Gal assays were performed with different ligand treatments and indicate that ING1b enhances the level of senescent cells without synergizing with AR . " p16//p27//AR Upregulation//--//-- Co-IP//Pull-down assay//Fluorescence microscopy "The results indicated that ING1b upregulates mRNA and protein expression levels of p16 in LNCaP-ING1b cells.Moreover, ING1b has an additive effect along with the AR antagonist Casodex on p16 mRNA induction. ING1b also stabilized p27 protein level in LNCaP-ING1b cells while not altering p27mRNA level (data not shown).In line with the previous results, AR protein was indeed detected in the immunoprecipitates with anti-ING1 antibody whereas no AR was detected in the negative control using a nonspecific antibody.AR protein could be detected with GST-ING1b eluates whereas the negative GST control did not show AR signal. Quantitative colocalization analyses of the images from cotransfected cells suggest that ING1b colocalizes with AR in the nucleus excluding the nucleolus with a high statistical significance." -- -- -- -- Human HL cellular senescence 26993046 Sen_G_0125 TGFB1 7040 protein coding HBEC -- Idiopathic pulmonary fibrosis Accelerate SA-β-gal activity assay//Flow cytometry//RT-PCR//Western blot "TGF-β1 increased the percentage of SA-β-gal staining cells and concomitantly induced the expression of p21/waf-1, as assessed by RT-PCR and Western blotting . Cell cycle analysis also showed an increase of senescent cells, as reflected by an accumulation of cells in the G0/G1phase . Furthermore, the accumulation of cells in the G0/G1phase was slightly increased after an additional 48-h incubation without TGF-β, suggesting irreversible cell cycle arrest after TGF-β treatment." p21 Upregulation SA-β-gal activity assay//Flow cytometry//Western blot "TGF-β1 increased the percentage of SA-β-gal staining cells and concomitantly induced the expression of p21/waf-1, as assessed by RT-PCR and Western blotting." -- -- -- -- Human L cellular senescence 21224216 Sen_G_0126 SIRT6 51548 protein coding HBEC -- Idiopathic pulmonary fibrosis Prevent SA-β-gal activity assay "Overexpression of SIRT6 significantly suppressed the percentage of SA-β-gal staining cells after TGF-β1 treatment. In contrast, SIRT6 siRNA dramatically increased the percentage of SA-β-gal staining cells, indicating that TGF-β-induced intrinsic SIRT6 upregulation alone is not sufficient to completely inhibit senescence, but SIRT6 has the ability to antagonize TGF-β-induced cellular senescence in HBEC." TGF-β Downregulation SA-β-gal activity assay "Overexpression of SIRT6 significantly suppressed the percentage of SA-β-gal staining cells after TGF-β1 treatment. In contrast, SIRT6 siRNA dramatically increased the percentage of SA-β-gal staining cells, indicating that TGF-β-induced intrinsic SIRT6 upregulation alone is not sufficient to completely inhibit senescence, but SIRT6 has the ability to antagonize TGF-β-induced cellular senescence in HBEC." -- -- -- -- Human L cellular senescence 21224216 Sen_G_0127 SIRT1 23411 protein coding HDF -- Colorectal cancer Prevent SA-β-gal activity assay "Only in c-MYC immortalized but not in primary HDFs down-regulation of SIRT1 by siRNAs induced a pronounced increase in cell size and senescence-associated β-galactosidase activity at pH 6, two markers of cellular senescence." c-Myc -- Western blot HDFs immortalized by retroviral introduction of constitutive expression of c-MYC (6) showed increased expression of SIRT1 protein. -- -- -- -- Human HL cellular senescence 22190494 Sen_G_0128 DUSP3 1845 protein coding "HeLa,MeWo" -- Cervical cancer Prevent SA-β-gal activity assay The results indicated that decreasing endogenous DUSP3 activity per se caused increased cellular senescence of both cell lines after 72 h . -- -- -- -- -- -- -- -- Human L cellular senescence 28389334 Sen_G_0129 SIRT1 23411 protein coding Human Nucleus pulposus cell -- Disc degenerative disease Prevent Western blot//RT-PCR The expression of p16 and p21 mRNA were higher in the resveratrol group than the control; while their expression increased in the Sirt siRNA plasmid group .Western blotting results were consistent with that of the results of reverse transcription-qPCR. -- -- -- -- -- -- -- -- Human L cellular senescence 27792110 Sen_G_0130 SRSF2 6427 protein coding HAoEC -- Aging Prevent SA-β-gal activity assay//Knockdown Knockdown of either SRSF2 or HNRNPD expression was sufficient to cause an increase in the numbers of senescent cells from 4 % to 6 % and 63% for HNRNPD and SRSF2 respectively . -- -- -- -- -- -- -- -- Human HL cellular senescence 30026406 Sen_G_0131 HNRNPD 3184 protein coding HAoEC -- Aging Prevent SA-β-gal activity assay//Knockdown Knockdown of either SRSF2 or HNRNPD expression was sufficient to cause an increase in the numbers of senescent cells from 4 % to 6 % and 63% for HNRNPD and SRSF2 respectively . -- -- -- -- -- -- -- -- Human HL cellular senescence 30026406 Sen_G_0132 CSNK1A1 1452 protein coding IMR-90 -- Colorectal cancer Prevent SA-β-gal activity assay//Western blot//qRT-PCR "Likewise, ablation of Csnk1a1 in MEFs and mRNA depletion of the corresponding gene, CSNK1A1, in human primary fibroblasts (IMR-90 cells) resulted in a persistent DDR and a senescence phenotype." -- -- -- -- p53 -- Western blot//Immunohistochemistry "By contrast, Csnk1a1Dgut mice showed marked p53 expression. Transcriptome analysis revealed that, not only the Wnt cascade, but also the p53 pathway is strongly induced byCsnk1a1 ablation . This was confirmed by monitoring the induction of several specific p53 target genes, including the anti-proliferative gene p21." Human HL cellular senescence 21331045 Sen_G_0133 CSF2 1437 protein coding SH-SY5Y -- Aging Accelerate SA-β-gal activity assay//Western blot//qRT-PCR "Beta-Galactosidase Assay The average number of SA-β-gal-stained SH-SY5Y cells in the GM-CSF group was 4.75± 0.96, which was significantly higher than that in the control group." -- -- -- -- -- -- -- -- Human L cellular senescence 30807472 Sen_G_0134 FOXM1 2305 protein coding MEF -- Aging Prevent SA-β-gal activity assay "Moreover, significantly higher numbers of Foxm1-/- MEFs displayed SA-β-gal and senescent morphology compared with WT MEFs following exposure to 5 Gy of g-irradiation." NBS1//P-ATM//FHRE --// --// -- "Western blot//Luciferase reporter assay " "The result showed that overexpression of FOXM1 caused an induction of NBS1 levels and ATM phosphorylation in MCF-7 cells ,further confirming that FOXM1 regulates NBS1 expression and thus MRN complex formation to promote ATM activation and phosphorylation.We observed the (WT) NBS1 promoter activity was augmented by FOXM1 in a dose-dependent manner, whereas the mut NBS1 promoter had lower basal promoter activity and was not inducible by FOXM1." -- -- -- -- Human HL cellular senescence 24141789 Sen_G_0135 NBS1 821254 protein coding MCF-7 -- Aging Prevent Crystal violet assay//SA-β-gal activity assay "Like FOXM1, NBS1-knockdown sensitized MCF-7 and MCF-7EpiRcells to long-term proliferative arrest following treatment with epirubicin. Consistently, NBS1 knockdown in MCF-7 and MCF-7EpiRcells also enhanced the number of cells exhibiting SA-β-gal activity and morphology at 0 and 10nM epirubicin." -- -- -- -- -- -- -- -- Human HL cellular senescence 24141789 Sen_G_0136 SIRT1 23411 protein coding HUVEC -- Aging Prevent SA-β-gal activity assay Overexpression of Sirt1 and resveratrol decreased SA-βgal–positive cells and specific morphological senescent changes induced by sirolimus and everolimus. -- -- -- -- -- -- -- -- Human L cellular senescence 19520256 Sen_G_0137 SUMO2 6613 protein coding NPC -- Disc degenerative disease Accelerate SA-β-gal activity assay The positive rate of cell senescence in the model and NC groups were significantly higher than that in the sham group (p < 0.05). The positive rate of cell senescence in the shRNA group was significantly lower than that in the IDD group (p < 0.05). -- -- -- -- p53 -- qRT-PCR "The mRNA and protein levels of SUMO2, p53, p21, MDM2, GADD45a, MMP-2, HIF-1α, and the p53 phosphorylation level of NPCs in the shRNA group were significantly lower than those in the IDD group (p < 0.05), but the mRNA and protein levels of CDK2/4 and CyclinB1 were significantly higher than that in the IDD group (p < 0.05). There was no significant difference observed between the model and NC group ." Human L cellular senescence 29700214 Sen_G_0138 YAP1 10413 protein coding h-PDLSC -- Aging Prevent SA-β-gal activity assay "Staining results showed that the OE YAP group had a lower senescence rate than the OE NC group (P<0.01), which indicates that activated YAP postponed the senescence of h-PDLSCs." CDK6//Cyclin B1//P18//p27 Upregulation//Upregulation//Downregulation//Downregulation Western blot "In Western blotting, cyclin-dependent kinase 6 (CDK6) and cyclin B1 were upregulated, while CDK inhibitors P18 and P27 were downregulated when YAP was overexpressed. " ERK Upregulation Western blot "The expression of P-Msk1, which can phosphorylate ERK, increased when YAP was overexpressed. At the same time, the protein levels of P-ERK1/2 and its target proteins P-P90RSK and P-Msk1 increased in the OE YAP group." Human L cellular senescence 30123063 Sen_G_0139 CDKN1A 1026 protein coding MDA-MB-231 -- Cancer Prevent SA-β-gal activity assay//Knockdown "When senescence-associated β-galactosidase staining was performed, where only cells in senescence developed blue staining, the results showed that β-galactosidase activity was greatly increased by p21 knockdown." -- -- -- -- -- -- -- -- Human L cellular senescence 26187313 Sen_G_0140 CAV1 857 protein coding "HCT116,NIH-3T3" -- Aging Accelerate SA-β-gal activity assay//Western blot "We found that only the expression of the mutant form of Nrf2 that cannot bind to caveolin-1 and is constitutively Activate (Φ A-Nrf2) protected cells from oxidative stress induced premature senescence, as shown by reduced staining for senescence-associated β-galactosidase activity (SA-β-gal) and p21Waf1/Cip1 protein expression. The stable overexpression of caveolin-1 in HCT116 cells potentiated the oxidative stress–induced up-regulation of p21Waf1/Cip1 and p16 expression and promoted the development of SA-β-gal–positive cells." -- -- -- -- Nrf2 Binding SA-β-gal activity assay//Pull-down assay "We found that only the expression of the mutant form of Nrf2 that cannot bind to caveolin-1 and is constitutively Activate protected cells from oxidative stress induced premature senescence, as shown by reduced staining for senescence-associated β-galactosidase activity (SA-β-gal) and p21Waf1/Cip1 protein expression. We found that WT -Nrf2-His bound to caveolin 1 (residues 82–101)–GST." Human L cellular senescence 23637463 Sen_G_0141 GPX3 2878 protein coding "LO2,MIHA" -- Aging Prevent SA-β-gal activity assay "The cellular senescence of liver cells was induced using 500 M H2O2 treatment for 1 hour and then the liver cells were cultured with normal medium. Forty eight hours later, we found that recombinant GPx3 protein (rGPx3) significantly suppressed hepatic senescence in LO2 and MIHA cells in a dose dependent manner." CD44//NOX4//SERPINB2 Downregulation//Downregulation//Downregulation PCR array "48h after rGPx3 treatment, 4 down-regulated gene candidates (CD44, Nox4, IFNG, SERPINB2) were identified . Three of them (CD44, Nox4, SERPINB2) were significantly up-regulated when cellular senescence was established (after H2O2 treatment), and sharply decreased upon rGPx3 treatment." -- -- -- -- Human HL cellular senescence 29290803 Sen_G_0142 FOXM1 2305 protein coding H929 -- Multiple myeloma Prevent SA-β-gal activity assay//Knockdown Left shows that FOXM1 knockdown sufficed to induce β-gal activity in myeloma. Rb Binding Western blot "Densitometric analysis of Western blots showed that total Rb and phosphorylated Rb (pRb) were increased in FOXM1Hi cells by 20-40%, with somewhat higher levels seen in XG1 than CAG cells. Conversely, Rb and pRb were decreased in FOXM1Lo cells (~ 30 to 50%), with the loss of the latter somewhat exceeding that of the former." -- -- -- -- Human HL cellular senescence 30463534 Sen_G_0143 IL1B 3553 protein coding Osteoarthritic Chondrocyte -- Osteoarthritis Accelerate Flow cytometry//SA-β-gal activity assay "In preliminary experiments (data not shown), we also confirmed the IL-1β–induced premature senescence at several concentrations of IL-1β(0.1, 1.0, 5.0, and 10.0 ng/ml) in vitro. The results indicated that premature senescence of chondrocytes was induced by IL-1β at lower concentrations as well." Caveolin-1//p38MAPK//CII Upregulation//Upregulation//Downregulation qPCR//Western blot//qRT-PCR//ELISA "Quantitative real-time RT-PCR analysis showed that both IL-1β and H2O2 enhanced caveolin 1 mRNA expression in a time-dependent manner, with the peak level occurring at 3 hours after stimulation(n ＝ 3). By Western blot analysis, we confirmed that both IL-1β and H2O2 also induced prolonged up-regulation of caveolin 1 protein expression (n = 3).Expression levels of phospho–p38 MAPK were markedly increased after IL-1β stimulation, with a peak level reached at 12 hours and sustained for more than 24 hours, whereas levels of phospho–ERK-1/2 were transiently downregulated from 6 to 12 hours.Furthermore, pretreatment with the specific p38 MAPK inhibitor SB202190 blocked the enhancement of SA–β-gal activity induced by IL-1β and H2O2.Quantitative real-time RT-PCR revealed a marked reduction in levels of expression of mRNA for CII and aggrecan in chondrocytes 3 days after stress with IL-1β or H2O2. Decreased production of CII from senescent chondrocytes after administration of either stress factor was also confirmed by ELISA (n = 4)." -- -- -- -- Human L cellular senescence 16508959 Sen_G_0144 CAV1 857 protein coding Osteoarthritic Chondrocyte -- Osteoarthritis Accelerate SA-β-gal activity assay Pretreatment with caveolin 1 antisense ODN significantly inhibited the up-regulation of caveolin 1 expression induced by IL-1β and H2O2 (n = 4) and also significantly blocked the increase in SA–β-gal activity induced by IL-1β and H2O2 (n = 4). p38MAPK//CII Upregulation//Downregulation Western blot//qRT-PCR//ELISA "Compared with chondrocytes transfected with empty vectors, the chondrocytes transduced with caveolin 1 cDNA showed higher levels of phospho–p38 MAPK and lower levels of phospho–ERK-1/2. In addition, caveolin 1 overexpression impaired the ability of chondrocytes to express CII (n = 4) and aggrecan (n = 4)." -- -- -- -- Human L cellular senescence 16508959 Sen_G_0145 FN1 2335 protein coding "U251,T98G" -- Aging Prevent SA-β-gal activity assay//qRT-PCR//Knockdown "Compared to that in the blank and NC groups, the cell senescence rate was notably increased in the siRNA-FN1 group, and the expression levels of p16 and p21 were significantly elevated (all P < 0.05)." -- -- -- -- PI3K-Akt Activation Luciferase reporter assay//Western blot//qRT-PCR "Compared to the pGL3- basic group, the luciferase activity of the pGL3-FN1 group was increased in both the U251 and T98G cells. Compared to blank and NC groups, the expression levels of FN1, PI3K, and AKT were all decreased in U251 and T98G cells in the siRNA-FN1 group, but the expression level of PTEN increased." Human HL cellular senescence 30048971 Sen_G_0146 PTTG1 9232 protein coding "MCF-7,MCF 10A" -- Breast cancer Accelerate SA-β-gal activity assay "By measuring the activity of senescence-associated b-galactosidase (SA-β-Gal), we found that hPTTG1 overexpression significantly reinforced senescence in both cell lines." IL-8//GROa Upregulation//Upregulation Cytokine array//ELISA "With antibody arrays, we determined that hPTTG1 overexpression led to a considerable induction in IL-8 and GROa. Because the antibody arrays were semiquantitative, we performed ELISA (enzyme-linked immunosorbent assay) analyses to confirm this result." CXCR2-p21//NF-κB Upregulation//Activation Dual-Luciferase reporter assay//CHIP array "Indeed, in both cells, hPTTG1 overexpression increased the expression of the p53 and p21 . Furthermore, in hPTTG1-overexpressing MCF-7 cells, deletions of the NF-kB binding site eliminated the luciferase activity of the IL-8 and GROa promoters. These results were further confirmed by ChIP (chromatin immunoprecipitation) assay, which revealed that the binding capacities of p65 were enhanced by hPTTG1 overexpression ." Human HL cellular senescence 22789011 Sen_G_0147 SALL1 6299 protein coding "MCF-7,MDA,E0771" -- Breast cancer Accelerate SA-β-gal activity assay "We observed that transfection of SALL1 in MCF-7, MDA, and E0771 tumor cells significantly increased the number of SA-βGal+ cells, indicating the induction of tumor cell senescence." ATM//H2AX//53BP1//Chk2 --//--//--//-- Flow cytometry "Overexpression of SALL1 significantly induced Activate, phosphorylated ATM in MCF-7, MDA and E0771 cancer cells. We observed that transfection of SALL1, but not SALL4 or control vector also significantly induced phosphorylation of H2AX, 53BP1 and CHK2 in MCF-7, MDA and E0771 cells (Data not shown)." p38 MAPK//ERK1/2//mTOR --//--//Activation Western blot "We first determined the activation and phosphorylation of MAPKs, including ERK1/2, p38 and JNK in breast cancer cells transfected with SALL1 using western blot analyses. We found that transfection of SALL1 but not mutated SALL1 selectively activated ERK1/2 and p38, but not JNK, resulting in significantly enhanced phosphorylation of ERK1/2 and p38 in both MCF-7 and E0771 breast cancer cells. Transfection of SALL1 but not mutated SALL1 in both MCF-7 and E0771 breast tumor cells significantly induced the phosphorylation of mTOR, p70S6K, and 4E-BP1, further confirming the activation of mTOR signaling in tumor cells after SALL1 expression." Human HL cellular senescence 29625565 Sen_G_0148 DUSP1 1843 protein coding SO-Rb5 -- Aging Accelerate SA-β-gal activity assay//Knockdown "DUSP1 plasmid and siRNA transfection enhanced and attenuated SO-Rb5 cell senescence induced by AGII, respectively, revealing that DUSP1 participated in SO-Rb5 cell senescence induced by AGII." -- -- -- -- Akt Downregulation Western blot "DUSP1 plasmid and siRNA transfection inhibited and strengthened Akt signaling in SO-Rb5 cell senescence induced by AGII, respectively, revealing that DUSP1 participated in SO-Rb5 cell senescence induced by AGII." Human L cellular senescence 30536303 Sen_G_0149 CIT 11113 protein coding "Daoy ,ONS-76" -- Medulloblastoma Prevent SA-β-gal activity assay//Knockdown "In addition, CITK knocked-down cells had a senescent morphology. Accordingly, CITK shRNA induction led to strong increase of cells positive for β-galactosidase cytochemical reaction, which is typical of senescent cells." -- -- -- -- -- -- -- -- Human L cellular senescence 29921697 Sen_G_0150 KLF4 9314 protein coding "T-REx-293 KLF4,T-REx-HeLa KLF4" -- Aging Accelerate SA-β-gal activity assay//Western blot "Then we characterized senescence in KLF4 overexpressed cells. Senescenceassociated β-galactosidase activity (SA-β-gal) markedly increased in cells with KLF4 overexpression . We also detected the senescence related proteins by Western Blot, and found that p21W AF1/CIP1 protein was strongly accumulated after KLF4 overexpression, accompanied by reduced expression of Cyclin E1 and increased expression of Suv39H1, but no significant variation of p53 and p16Ink4a." p21 Upregulation CHIP//qPCR//Western blot "We found that p21 mRNA level was induced by KLF4 overexpression, and KLF4 could bind to the promoter region of p21 gene, confirmed by ChIP assay. When p21 protein was knocked down, KLF4 induction could induce only about 8 percent of senescent cells, comparing with more than 70% senescent cells in control cells ." miR-203-Survivin-p12 -- Western blot//qPCR//Luciferase reporter assay "Protein level of survivin and mRNA expression were both inhibited by KLF4 overexpression. Additionally, p21 upregulation induced by KLF4 was significantly inhibited.In our study, survivin protein could directly bind to the distal and proximal p53 binding sites of p21 promoter in T-REx-293 KLF4 cells, as confirmed by ChIP assay. T-REx-293 cells were co-transfected with survivin and reporter plasmids, and reporter assay showed that the transcription activities of both pGL3 p21 5′, pGL3 p21 3′ were significantly inhibited by survivin. ChIP assay showed that KLF4 could directly bind to -189bp to +11bp fragment of miR-203 gene. To test whether miR-203 can target survivin or not, pre-miR-203 precursor and miR-203 inhibitor were transfected into T-REx-293 cells, respectively.mRNA expression of survivin was inhibited by pre-miR-203, while inhibition of miR-203 could increase survivin expression, the similar result was shown in protein level." Human HL cellular senescence 27531889 Sen_G_0151 PIK3R4 30849 protein coding HUVEC -- Aging Prevent SA-β-gal activity assay "The β-galactosidase positive rates in cells of control, scrambled and Vps15-shRNA transfection groups were22.4±1.9%, 24.3±2.1% and 42.5±2.5%, respectively.Moreover, both the β-galactosidase positive- (23.1±2.7%) and apoptotic rates (10.4±1.1%) in cells with Vps15 reexpression were lower than in cells of Vps15 knockdown group." -- -- -- -- -- -- -- -- Human L cellular senescence 31356904 Sen_G_0152 NFKB2 4791 protein coding Primary Fibroblast -- Aging Prevent SA-β-gal activity assay "When analyzing siRNA depletion of NF-kB2 and RelB in NHD fibroblasts, we also observed that cells ceased to proliferate, changed morphology and after 7 days in culture, using the acidic β galactosidase assay, were found to enter a senescent state." p53//EZH2 --//Binding SA-β-gal activity assay//Knockdown//CHIP-Seq "Importantly induction of senescence and ROS upon depletion of NF-kB2, RelB and Bcl-3 was p53 dependent.This result was confirmed by mining of ChIP-Seq data from GM12878 B-cells [40], where binding of all NF-kB subunits to the EZH2 promoter was seen." -- -- -- -- Human HL cellular senescence 25255445 Sen_G_0153 ATM 472 protein coding HCT116 -- Colorectal cancer Accelerate SA-β-gal activity assay//Flow cytometry "We used a widely known inhibitor of the PIKKs—caffeine20, and analyzed different markers of cell senescence. Higher SA-β-Gal activity and cell granularity, measured by flow cytometry, were observed in dox-treated cells than in control (untreated) ones, while pretreatment with caffeine substantially decreased the activity of SA-β-Gal and cell granularity and preserved the cell proliferation capabilities." -- -- -- -- -- -- -- -- Human L cellular senescence 29352261 Sen_G_0154 ATR 545 protein coding HCT116 -- Colorectal cancer Accelerate SA-β-gal activity assay//Flow cytometry "We used a widely known inhibitor of the PIKKs—caffeine20, and analyzed different markers of cell senescence. Higher SA-β-Gal activity and cell granularity, measured by flow cytometry, were observed in dox-treated cells than in control (untreated) ones, while pretreatment with caffeine substantially decreased the activity of SA-β-Gal and cell granularity and preserved the cell proliferation capabilities." -- -- -- -- -- -- -- -- Human L cellular senescence 29352261 Sen_G_0155 PRKDC 5591 protein coding HCT116 -- Colorectal cancer Accelerate SA-β-gal activity assay//Flow cytometry "We used a widely known inhibitor of the PIKKs—caffeine20, and analyzed different markers of cell senescence. Higher SA-β-Gal activity and cell granularity, measured by flow cytometry, were observed in dox-treated cells than in control (untreated) ones, while pretreatment with caffeine substantially decreased the activity of SA-β-Gal and cell granularity and preserved the cell proliferation capabilities." -- -- -- -- -- -- -- -- Human L cellular senescence 29352261 Sen_G_0156 CHUK 1147 protein coding -- Tumor tissue Lung cancer Accelerate SA-β-gal activity assay//Ki67 staining KrasG12D;IkkαΔLu lung ADCs displayed substantially less SA-β-gal staining and more Ki67 than KrasG12D ADCs. -- -- -- -- Nrf2-NQO1 Downregulation SA-β-gal activity assay//Western blot "Indeed, KrasIKKαL cells expressed reduced IKKα, NRF2, NQO1, p53, and p21 and showed attenuated cell senescence compared with Kras-CL cells." Human HL cellular senescence 29311298 Sen_G_0157 PTEN 5728 protein coding MCF-7 -- Aging Prevent Western blot//Immunocytochemistry//SA-β-gal activity assay PTEN depleted MCF7 cells displayed prematurely senescent phenotypes in a p53/p21-dependent manner.The SA-β-Gal positivity of PTEN KO MEFs was dramatically attenuated in Torin1-treated cells compared to Rapa-treated cells. mTORC1//mTORC2//p53 --//--//Activation Western blot//IP "The activities of both mTORC1 and mTORC2 were increased along with that of AKT during PICS.Indeed, p53 phosphorylation at S15 was abolished in PTEN-depleted cells expressing mTOR KD. We further conducted an immunoprecipitation (IP) assay with anti-p53, and observed endogenous association of mTOR with p53 during PICS . Raptor and Rictor were also found in the p53 immunoprecipitates, indicating that both mTORC1 and mTORC2 physically associated with p53." -- -- -- -- Human L cellular senescence 30337688 Sen_G_0158 TP53 7157 protein coding "U87,U2OS" -- Aging Accelerate SA-β-gal activity assay//EdU assay//Immunofluorescence "Nutlin-3a treatment strikingly induced cellular senescence in U87 and U2OS cells, as shown by positive senescence-associated β-galactosidase (SA-β-gal) staining, reduction of lamin B1, and reduced 5-ethynyl-2 -deoxyuridine (EdU) incorporation, p16." EGFR//DYRK1A Downregulation//Downregulation Western blot//SA-β-gal activity assay "Indeed, ectopic expression of EGFR in U87 or U2OS cells significantly attenuated senescence induced by p53 activation.Activation of p53 by Nutlin-3a led to a significant reduction in DYRK1A." EGFR-MEK-ERK Downregulation Western blot "Indeed, the level of phosphorylated (Activate) form of ERK was decreased in Nutlin-3a treated U87 and U2OS cells." Human L cellular senescence 30910997 Sen_G_0159 POLE2 5427 protein coding Fibroblast -- Aging Prevent Cell counting//EdU assay//SA-β-gal activity assay//RT-qPCR//Knockdown "To this end, we tested several DNA repair genes by knocking-down their expression in human fibroblasts. Strikingly, knock-down of POLE2, BARD1, FEN1, RAD51, EXO1, BRCA1, or BLM alone was able to induce hallmarks of premature senescence." -- -- -- -- p53-p21-Rb -- qRT-PCR "Furthermore, Nutlin-3 or AT7519 notably repressed DNA repair gene expression through a P53/ P21/RB pathway." Human HL cellular senescence 29449545 Sen_G_0160 BARD1 580 protein coding Fibroblast -- Aging Prevent Cell counting//EdU assay//SA-β-gal activity assay//RT-qPCR//Knockdown "To this end, we tested several DNA repair genes by knocking-down their expression in human fibroblasts.Strikingly, knock-down of POLE2, BARD1, FEN1, RAD51, EXO1, BRCA1, or BLM alone was able to induce hallmarks of premature senescence." -- -- -- -- p53-p21-Rb -- qRT-PCR "Furthermore, Nutlin-3 or AT7519 notably repressed DNA repair gene expression through a P53/ P21/RB pathway." Human HL cellular senescence 29449545 Sen_G_0161 FEN1 2237 protein coding Fibroblast -- Aging Prevent Cell counting//EdU assay//SA-β-gal activity assay//RT-qPCR//Knockdown "To this end, we tested several DNA repair genes by knocking-down their expression in human fibroblasts. Strikingly, knock-down of POLE2, BARD1, FEN1, RAD51, EXO1, BRCA1, or BLM alone was able to induce hallmarks of premature senescence." -- -- -- -- p53-p21-Rb -- qRT-PCR "Furthermore, Nutlin-3 or AT7519 notably repressed DNA repair gene expression through a P53/ P21/RB pathway." Human HL cellular senescence 29449545 Sen_G_0162 RAD51 5888 protein coding Fibroblast -- Aging Prevent Cell counting//EdU assay//SA-β-gal activity assay//RT-qPCR//Knockdown "To this end, we tested several DNA repair genes by knocking-down their expression in human fibroblasts . Strikingly, knock-down of POLE2, BARD1, FEN1, RAD51, EXO1, BRCA1, or BLM alone was able to induce hallmarks of premature senescence." -- -- -- -- p53-p21-Rb -- qRT-PCR "Furthermore, Nutlin-3 or AT7519 notably repressed DNA repair gene expression through a P53/ P21/RB pathway." Human HL cellular senescence 29449545 Sen_G_0163 EXO1 9156 protein coding Fibroblast -- Aging Prevent Cell counting//EdU assay//SA-β-gal activity assay//RT-qPCR//Knockdown "To this end, we tested several DNA repair genes by knocking-down their expression in human fibroblasts. Strikingly, knock-down of POLE2, BARD1, FEN1, RAD51, EXO1, BRCA1, or BLM alone was able to induce hallmarks of premature senescence." -- -- -- -- p53-p21-Rb -- qRT-PCR "Furthermore, Nutlin-3 or AT7519 notably repressed DNA repair gene expression through a P53/ P21/RB pathway." Human HL cellular senescence 29449545 Sen_G_0164 BRCA1 672 protein coding Fibroblast -- Aging Prevent Cell counting//EdU assay//SA-β-gal activity assay//RT-qPCR "To this end, we tested several DNA repair genes by knocking-down their expression in human fibroblasts. Strikingly, knock-down of POLE2, BARD1, FEN1, RAD51, EXO1, BRCA1, or BLM alone was able to induce hallmarks of premature senescence." -- -- -- -- p53-p21-Rb -- qRT-PCR "Furthermore, Nutlin-3 or AT7519 notably repressed DNA repair gene expression through a P53/ P21/RB pathway." Human HL cellular senescence 29449545 Sen_G_0165 BLM 641 protein coding Fibroblast -- Aging Prevent Cell counting//EdU assay//SA-β-gal activity assay//RT-qPCR//Knockdown "To this end, we tested several DNA repair genes by knocking-down their expression in human fibroblasts. Strikingly, knock-down of POLE2, BARD1, FEN1, RAD51, EXO1, BRCA1, or BLM alone was able to induce hallmarks of premature senescence." -- -- -- -- p53-p21-Rb -- qRT-PCR "Furthermore, Nutlin-3 or AT7519 notably repressed DNA repair gene expression through a P53/ P21/RB pathway." Human HL cellular senescence 29449545 Sen_G_0166 SIX1 6495 protein coding IMR-90 -- Aging Prevent SA-β-gal activity assay//Cell morphological analysis//BrdU assay//Western blot "Stable silencing of SIX1 in early passage IMR90 cells markedly reduced proliferation, as assessed by bromodeoxyuridine incorporation.Most importantly, IMR90 shSIX1 cells displayed features of cellular senescence, including senescent morphology with extended vacuolized cytoplasms and prominent nucleoli, and increased number of senescence-associated β-galactosidase-positive cells." p16//Polycomb --//-- Western blot//Immunofluorescence//qPCR//CHIP "In agreement with the inverse correlation between SIX1 and p16 found in ER:Ras induced senescence, we observed that p16 protein and RNA levels were significantly increased in shSIX1-IMR90 cells, as did the number of p16-positive cells in immunofluorescence assays.To test if SIX1 could cooperate with Polycomb complexes in senescence, we analyzed changes in the Polycomb-associated mark H3K27Me3. ChIP showed a clear reduction of H3K27Me3 in the INK4A promoter (~5-fold) in shSIX1-senescent cells , in the absence of gross changes in the H3K27Me3 nuclear level or distribution, similar to other senescence stimuli." -- -- -- -- Human HL cellular senescence 26500063 Sen_G_0167 PIM1 5292 protein coding 2BS -- Aging Accelerate SA-β-gal activity assay//SAHF//Knockdown "Furthermore, PIM-1 knockdown resulted in a reduced percentage of cells with senescence-associated b-galactosidase (SA-β-gal) staining and SAHF formation, two well-known markers of cellular senescence." HP1γ -- IP//Western blot//qPCR "IP with anti-PIM-1 antibody successfully pulled down HP1γ, while reciprocal experiment using anti-HP1γ antibody validated the interaction between PIM-1 and HP1γ in senescent cells." IL-6-STAT3 -- Luciferase reporter assay//Knockdown "Reporter analysis showed that RasV12 overexpression increased the wild-type PIM-1 promoter activity, and mutation of the STAT3 binding site dramatically attenuated the RasV12- induced promoter activity .In addition, expressing IL-6 shRNA suppresses the PIM-1 promoter activity." Human L cellular senescence 25040935 Sen_G_0168 GSK3A 2931 protein coding Chang -- Aging Prevent SA-β-gal activity assay//Cell morphological analysis "As expected, at the subcytotoxic doses of SB415286, cells acquired senescence phenotypes in dose- and time-dependent manners, as shown by the gain of SA-β-gal activity and flattened, enlarged cellular morphology ." GS -- Western blot//Cell viability assay//SA-β-gal activity assay "GS phosphorylation was decreased when SB415286, a small molecule inhibitor of GSK3 (Coghlan et al., 2000), was applied to Chang cells at concentrations < 15 g mL 1, but rather increased at higher concentrations of SB415286 (20 and 25 g mL 1).Clearly, GS was activated by dephosphorylation in these conditions, resulting in the progressive accumulation of glycogen along with the senescence phenotypes, such as SA-β gal and induction of p16 and p21." -- -- -- -- Human L cellular senescence 18782348 Sen_G_0169 GSK3B 2932 protein coding Chang -- Aging Prevent SA-β-gal activity assay//Cell morphological analysis "As expected, at the subcytotoxic doses of SB415286, cells acquired senescence phenotypes in dose- and time-dependent manners, as shown by the gain of SA-β-gal activity and flattened, enlarged cellular morphology." GS -- Western blot//Cell viability assay//SA-β-gal activity assay "GS phosphorylation was decreased when SB415286, a small molecule inhibitor of GSK3 (Coghlan et al., 2000), was applied to Chang cells at concentrations < 15 g mL 1, but rather increased at higher concentrations of SB415286 (20 and 25 g mL 1).Clearly, GS was activated by dephosphorylation in these conditions, resulting in the progressive accumulation of glycogen along with the senescence phenotypes, such as SA-β gal and induction of p16 and p21." -- -- -- -- Human L cellular senescence 18782348 Sen_G_0170 EZH2 2146 protein coding "MKN28,SGC-7901" -- Aging Prevent SA-β-gal activity assay//Cell morphological analysis "In MKN28 and SGC7901 cells, a series of phenotypic changes which are the hallmarks of cellular senescence were observed after EZH2 shRNA treated, including flat and enlarged morphology and an increase in SA-β- gal activity but not in AGS cells." H3K27me3//INK4/ARF//p14//p15INK4b//p16 --//--//--//--//-- Western blot//qRT-PCR "Moreover, a significant decrease of H3K27me3 was also observed compared with negative control.Western blot analysis confirmed the activation of INK4/ARF locus leading by EZH2 depletion at protein level.For cells with EZH2 down-regulated, p14ARF, p15INK4b and p16INK4a transcript levels were increased dramatically compared with control cells ." p15-Rb -- Western blot "Meanwhile, depletion of EZH2 increased p15INK4b expression dramatically, demonstrating that EZH2 blocks p15INK4b activation and this regulation maybe make great contribution to cellular senescence induced by EZH2 silencing in MNK28 cells. Moreover, the decreased p15INK4b protein expression was associated with increased hyperphosphorylation of its downstream, Rb." Human L cellular senescence 26004298 Sen_G_0171 PEBP1 5037 protein coding A549 -- Aging Accelerate SA-β-gal activity assay//Western blot Elimination of RKIP could increase ERK activation and suppress the cellular senescence. p53 -- CHIP "Moreover, RKIP was a direct target of p53." Raf-MAPK Downregulation SA-β-gal activity assay//Western blot "Elimination of RKIP could increase ERK activation and suppress the cellular senescence. In contrast, suppression of Ras-Raf-ERK pathway using Raf kinase inhibitor could induce the cellular senescence ." Human L cellular senescence 23814485 Sen_G_0172 EGF 1950 protein coding HME -- Aging Prevent SA-β-gal activity assay Further analysis of HME cells cultured in the absence of EGF revealed that these cells displayed high SA-β-gal activity in addition to severely impaired proliferation . -- -- -- -- -- -- -- -- Human L cellular senescence 25367123 Sen_G_0173 IFNA1 3439 protein coding HDF -- Aging Accelerate SA-β-gal activity assay "Consistent with these observations, the knockdown of IFNα/βRα subunit expression decreased the number of SA-β-gal positive cells, which is indicative of the onset of cellular senescence in HDFs." IFI16//IL-1β//AIM2 --//Upregulation//-- Western blot "Similarly, the knockdown reduced basal and IFN-β induced levels of IFI16 protein.Interestingly, the treatment also increased levels of the cleaved IL-1β, a proinflammatory cytokine, which is indicative of the activation of an inflammasome.The knockdown of AIM2 expression in BPH-1 cells increased steady-state levels of STAT1 and potentiated IFN-g induced activation of STAT1. Importantly, the knockdown increased basal and the IFNg induced levels of the IFI16 protein." -- -- -- -- Human L cellular senescence 21471287 Sen_G_0174 IFNB1 3456 protein coding HDF -- Aging Accelerate SA-β-gal activity assay//Knockdown "Consistent with these observations, the knockdown of IFNα/βRα subunit expression decreased the number of SA-β-gal positive cells, which is indicative of the onset of cellular senescence in HDFs." IFI16//IL-1β//AIM2 --//Upregulation//-- Western blot "Similarly, the knockdown reduced basal and IFN-β induced levels of IFI16 protein. Interestingly, the treatment also increased levels of the cleaved IL-1β, a proinflammatory cytokine, which is indicative of the activation of an inflammasomeThe knockdown of AIM2 expression in BPH-1 cells increased steady-state levels of STAT1 and potentiated IFN-g induced activation of STAT1. Importantly, the knockdown increased basal and the IFNg induced levels of the IFI16 protein." -- -- -- -- Human L cellular senescence 21471287 Sen_G_0175 CSN2 1447 protein coding MEF -- Aging Accelerate Cell proliferation assay Expression of these antisense constructs partially bypasses growth arrest in senescence and is able to produce a moderate increase in the lifespan of MEFs. -- -- -- -- -- -- -- -- Human HL cellular senescence 17968325 Sen_G_0176 BRF1 2972 protein coding MEF -- Aging Accelerate Cell proliferation assay Expression of these antisense constructs partially bypasses growth arrest in senescence and is able to produce a moderate increase in the lifespan of MEFs. -- -- -- -- -- -- -- -- Human HL cellular senescence 17968325 Sen_G_0177 CDK4 1019 protein coding MCF-7 -- Aging Accelerate SA-β-gal activity assay "MCF7 cells arrested for 6 days were stained for β-galactosidase and an increase in β-gal+ cells was detected in the doxycycline + PD– treated cells, suggesting that these nonproliferative cells were actually senescent and could not reenter cell cycle." -- -- -- -- -- -- -- -- Human L cellular senescence 29330290 Sen_G_0178 EGFR 1956 protein coding A549 -- Aging Accelerate SA-β-gal activity assay//Western blot "Examination of p53 wild-type cells revealed several features of senescence, including morphologic characteristics indicating premature differentiation and expression of senescence-associated β-galactosidase as well as increased levels of trimethylated histone H3K9 ." p53 -- SA-β-gal activity assay//Western blot "Examination of p53 wild-type cells revealed several features of senescence, including morphologic characteristics indicating premature differentiation and expression of senescence-associated β-galactosidase as well as increased levels of trimethylated histone H3K9." MEK-ERK -- Cell viability assay//Cell Proliferation assay//SA-β-gal activity assay "Using A549, ABC1, and HCC44 cells as representative examples, we observed that treatment with a MEK inhibitor indeed increased the fraction of cells with residual g-H2AX foci by 8.2% to 18.1%, which was comparable with the effects of EGFR inhibition in A549 cells. MEK inhibition also caused p21 induction, radiosensitization, cellular senescence, and did not further enhance the radiosensitizing effects of erlotinib,implicating the MEK–ERK pathway as one common effector pathway of radioresistance downstream of EGFR." Human L cellular senescence 21852385 Sen_G_0179 HRAS 3265 protein coding MEF -- Aging Accelerate SA-β-gal activity assay//Western blot "As expected, early-passage primary MEFs expressing H-RasV12 and WT KSR1 exhibited characteristics of cellular senescence, such as reduced proliferation in culture and SA β-galactosidase activity, concomitant with the induction of p16INK4a, p15INK4b, and p53 and sustained ERK activity." KSR1 -- SA-β-gal activity assay//Western blot "Early-passage primary MEFs expressing H-RasV12 and WT KSR1 exhibited characteristics of cellular senescence, such as reduced proliferation in culture and SA β-galactosidase activity, concomitant with the induction of p16INK4a, p15INK4b, and p53 and sustained ERK activity.However, MEFs expressing H-RasV12 and KSR1.CBM did not undergo proliferation arrest or demonstrate SA β-galactosidase activity, similar to primary MEFs expressing only H-RasV12." -- -- -- -- Human L cellular senescence 25002533 Sen_G_0180 KIR2DL4 3805 protein coding NK -- Aging Accelerate qRT-PCR "To test if TAK1 was required for the 2DL4-mediated SASP of resting NK cells, we stimulated resting NK cells with agonist Ab to 2DL4 for 16 h in the presence or absence of 1 mM TAK1 inhibitor. Quantitative RT-PCR was used to test the relative expression of a set of 10 genes that contribute to the senescent signature of primary NK cells. Upregulation of each of these 10 genes upon stimulation of NK cells with agonist Ab to 2DL4 was blocked in the presence of 1 mM TAK1 inhibitor . These results suggest that TAK1 is required for the SASP that results from the activation of primary NK cells by 2DL4." TAK1 -- qRT-PCR "To test if TAK1 was required for the 2DL4-mediated SASP of resting NK cells, we stimulated resting NK cells with agonist Ab to 2DL4 for 16 h in the presence or absence of 1 mM TAK1 inhibitor. Quantitative RT-PCR was used to test the relative expression of a set of 10 genes that contribute to the senescent signature of primary NK cells. Upregulation of each of these 10 genes upon stimulation of NK cells with agonist Ab to 2DL4 was blocked in the presence of 1 mM TAK1 inhibitor. These results suggest that TAK1 is required for the SASP that results from the activation of primary NK cells by 2DL4." -- -- -- -- Human L cellular senescence 24337384 Sen_G_0181 IGFBPL1 347252 protein coding HEC-1A -- Endometrial cancer Accelerate SA-β-gal activity assay//Western blot "SA-β-galactosidase staining, a golden standard for cellular senescence, indicated that HEC-1A-IGFBP-rP1 cells had a higher proportion of SA-β-galactosidase positive cells than the control cells. On the contrary, SA-β-galactosidase activity significantly decreased in Ishikawa-siRNA (HEC-1A-IGFBP-rP1) cells compared with negative controls . Moreover, p-RB, a key regulator in cellular senescence, was significantly reduced in HEC-1A-IGFBP-rP1 cells than control cells while other senescence-related proteins p21. p53 and p16, increased ~four-fold. 55-fold and 24-fol in HEC-1A-IGFBP-rP1, respectively. In contrast, Ishikawa-siRNA (IGFBP-rP1) cells presented a 2.8-fold increased expression of p-RB (P<0.01) and a decrease level of p16, p21 and p53 protein than control cells." -- -- -- -- ERK Downregulation SA-β-gal activity assay HEC-1A-IGFBP-rP1 cells with PD98059 presented more SA-β-galactosidase positive cells than HEC-1A with PD98059 treatment. Human L cellular senescence 29067463 Sen_G_0182 PATZ1 23598 protein coding HUVEC -- Aging Prevent SA-β-gal activity assay//Flow cytometry//BrdU assay//Knockdown "Knockdown of PATZ1 in young cells increased SA-β-gal staining activity.Although cell populations in S and G2/M phases were reduced, cells in G0/G1 increased in PATZ1 siRNA cells, suggesting that PATZ1 knockdown induces G0/G1 cell cycle arrest.Upregulation of PATZ1 in old cells increased cell proliferation and repressed SA-β-gal activity.PATZ1 overexpression also released old cells from G2/M cell cycle arrest, confirmed by increases in G1/G0 and S-cell populations as visualized by flow cytometry." -- -- -- -- p53 -- SA-β-gal activity assay//Knockdown "Downregulation of PATZ1 inhibited cell proliferation in p16INK4a-knockdown cells, but not in p53-knockdown cells.Transfection with PATZ1 siRNA didn't increase SA-β-gal activity in p53-knockdown cells ." Human HL cellular senescence 22052190 Sen_G_0183 SIRT1 23411 protein coding "SK-Hep-1,HepG2" -- Hepatocellular carcinoma Prevent Knockdown//Colony formation assay//SA-β-gal activity assay//Flow cytometry "Knockdown of SIRT1 reduced the number and size of SK-Hep-1 cell colonies .Cell-cycle analysis showed that significant G1arrest was observed in SK-Hep-1 and HepG2 cells. We also observed that gene silencing of SIRT1 in SK-Hep1 and HepG2 (p53 wild-type) resulted in cells that were enlarged in size, flattened in shape, and highly positive for SA-β-gal staining." -- -- -- -- -- -- -- -- Human HL cellular senescence 21527554 Sen_G_0184 AKR1B1 231 protein coding Keratinocyte -- Aging Prevent SA-β-gal activity assay//Knockdown "In cells transfected with siRNA against AR or treated with EBPC, this increase in SA β-Gal-positive cells was further enhanced." p53 -- Knockdown//Western blot "SiRNA-mediated knockdown of AR further increased the elevated expression of p53 induced by UVB radiation, indicating that AR prevents cellular senescence through the regulation of p53 protein expression." -- -- -- -- Human L cellular senescence 21182935 Sen_G_0185 SP1 6667 protein coding 2BS -- Aging Accelerate Knockdown//SA-β-gal activity assay "The results showed that Sp1-overexpressed cells were strongly stained blue versus the control. However, there were only a few dispersed cells that were SA-β-Gal-stained in the Sp1 knocked-down cells." p16 -- Luciferase reporter assay//Western blot "As determined by luciferase activity, Sp1 activated the p16INK4a promoter in a dose-dependent manner in both young and senescent 2BS cells,the p16INK4a promoter activity was inhibited by MTR in a dose-dependent manner in both young and senescent 2BS cells. Further, the p16INK4a expression was also reduced at mRNA and protein levels in 2BS cells, by 66% (mRNA level) and 48% (protein level) respectively, in the senescence group with 24 hours of MTR treatment.Western Blot showed that si-Sp1 remarkably reduced the expression of Sp1, which in turn lead to a reduction of p16INK4a expression." -- -- -- -- Human L cellular senescence 17225865 Sen_G_0186 ID1 3397 protein coding NPTX -- Aging Prevent SA-β-gal activity assay//Flow cytometry "The AS Id-1 clones displayed several morphological changes compared with the parental NPTX cells, characterized by enlarged, vacuolized and flattened cell bodies and loss of light refractability.The SA-β-gal staining further confirmed that AS Id-1 clones contained significantly more senescent cells .In addition, inactivation of Id-1 resulted in a decrease in S-phase cells , and an increase in G2/M phase cells." p21/WAF1//p27 --//-- Western blot//RT-PCR "Compared with the pBabe control, AS Id‐1 clones had much lower Id‐1 expression, along with increased p21WAF1/Cip1 and p27KIP1 protein levels.In agreement with the Western‐blot results, the addition of TGF‐β1 had a positive effect on p21WAF1/Cip1 and p27KIP1 mRNA expression and a negative effect on Id‐1 mRNA expression in AS Id‐1 clones." -- -- -- -- Human L cellular senescence 16686600 Sen_G_0187 TBX2 6909 protein coding B16 -- Melanoma Prevent SA-β-gal activity assay//Flow cytometry "Activation of ER-dnTbx2 by 4-OHT induces a marked accumulation of cells staining positive for SAβ-gal activity, ~25% of clone 1 B16 HA-ER-dnTbx2-expressing cells and ~80% of the clone 2 cells." p21 Upregulation Western blot "Western blot analysis using anti-p21 antibodies revealed that in cells expressing HA-ER, p21 expression was barely detectable either in the presence or absence of 4-OHT.The addition of 4-OHT to the HA-ER-dnTbx2-expressing cells led to a dramatic increase in p21 expression consistent with it acting to displace the endogenous Tbx2 from the p21 promoter.with both siRNAs efficiently down-regulating Tbx2 and consequently up-regulating p21 expression comparedwith the control siRNA ." -- -- -- -- Human L cellular senescence 15781639 Sen_G_0188 TNF 7124 protein coding HUVEC -- Aging Accelerate Flow cytometry//BrdU assay//Western blot//SA-β-gal activity assay "Cell cycle analysis revealed prolonged exposure to TNFα significantly impaired proliferation of cells, as evidenced by progressive decline in abundance of BrdU-incorporating cells accompanied by a decreased percentage of Sphase and increased G1/G2-phase cells compared to control, indicating proliferative arrest.Cells exposed to TNFα also developed classic senescent phenotypes, including a flattened, multi-nucleated, giant cell morphology, and concomitantly increased expression of senescence markers, including p16, p21, and cellular SA-β-gal activity." -- -- -- -- JAK-STAT Activation SA-β-gal activity assay//Western blot "The abundance of serine-phosphorylated STAT1 in cells undergoing senescence due to TNFα was sustained at day 3 and remained elevated at day 6, concomitant with the establishment of senescence .The phosphorylated forms of STAT1 and STAT3 peaked early and then slowly decayed in cells treated with conditioned medium compared to control." Human HL cellular senescence 29176033 Sen_G_0189 NOX4 50507 protein coding VSMC -- Aging Prevent SA-β-gal activity assay//BrdU assay "The number of SA-β-gal-positive cells correlated inversely with the level of NOX4 expression obtained after transfection with different siRNA sequences.Moreover, cells with diminished NOX4 expression cease to proliferate since a significant decrease in the number of cells able to replicate DNA was observed already 3 days after transfection. These cells did not restart DNA replication during the next 4 days of the culture." -- -- -- -- -- -- -- -- Human L cellular senescence 27655718 Sen_G_0190 BMI1 648 protein coding UCB-MSC -- Aging Prevent Western blot//SA-β-gal activity assay//MTT assay "The overexpression of BMI1 and the decreased expression of p16INK4a was confirmed via western blotting. BMI1-overexpressing hUCB-MSCs showed a reduced SA-β-gal activity compared to that of the control GFP-transduced cells.The depletion of BMI1 increased SA-β-gal staining and p16INK4a expression, showing the accelerated aging of these hUCB-MSCs. Cellular proliferation was also decreased in shBMI1-MSCs." -- -- -- -- -- -- -- -- Human L cellular senescence 27454161 Sen_G_0191 BAP1 8314 protein coding HeLa-S3 -- Cervical cancer Accelerate SA-β-gal activity assay "Strikingly, retroviral overexpression of BAP1 reduced cell proliferation and induced senescence-associated β-galactosidase (SA-β-gal) activity." ASXL1/2 -- Western blot "We found that BAP1 protein levels increased with ASXL1/2 expression in a dose-dependent manner. Conversely, ASXL1/2 protein levels were also increased following overexpression of BAP1." p53-p21 -- Western blot "We found that although the effect of BAP1 was less pronounced than the BAP1C91S?form, overexpression of this DUB induced the p53/p21 tumor suppressor pathway.Overexpression of BAP1ΔCTD, BAP1R666-H669, BAP1C91S-ΔCTD, or BAP1C91S-R666-H669?did not up-regulate p53/p21 indicating the requirement for ASXL1/2 in BAP1-mediated senescence ." Human L cellular senescence 26416890 Sen_G_0192 SOX1 6656 protein coding HONE1 -- Nasopharyngeal carcinoma Accelerate SA-β-gal activity assay//Flow cytometry We also found that overexpression of SOX1 in HONE1 cells enhanced SA-β-gal staining.Ectopic expression of SOX1 increased the number of cells in the G1/G0 phase (from 67.77?±?0.9% to 71.82?±?1.05%) and decreased the number of cells in the G2/M phase (from 12.7?±?0.10% to 9.3?±?0.10%) in HONE1 cells. c-Myc//Cyclin D1//p21//p27//Cyclin E Downregulation//Downregulation//Upregulation//Upregulation//Downregulation Western blot "A decrease in both c-Myc and Cyclin D1 protein was detected in SOX1-overexpressing HONE1 cells.In HONE1 cells, SOX1 overexpression significantly enhanced the expression of p21 and p27 but suppressed the expression of Cyclin E." Wnt-β-catenin Downregulation Western blot//Knockdown "Furthermore, the down-regulation of β-catenin induced by SOX1-overexpression was reversed by knockdown of SOX1 using siRNA. Consistently, β-catenin was down-regulated in a dose-dependent manner with increasing amounts of SOX1." Human L cellular senescence 25427424 Sen_G_0193 CDCA2 157313 protein coding IMR-90 -- Aging Accelerate SA-β-gal activity assay "CDCA2 over-expressing cells finally arrested their growth and showed SA-β-gal accumulation, just like control cells." SAmiR-494 Downregulation Luciferase reporter assay//Western blot "In the case of CDCA2 and ID4 the reporter gene expression was significantly reduced by SAmiR-494 or SAmiR-486-5p pre-miR transfection, respectively, with a variation of relative luciferase expression similar to the positive control OLFM4 (N).We also investigated the expression profiles of the two targets by Western blot analysis, upon SAmiRs over-expression or down-regulation in PDL 33 IMR90 cells.The decrease in CDCA2 and ID4 endogenous expression levels was well detectable at protein level." -- -- -- -- Human HL cellular senescence 24905922 Sen_G_0194 RBP2 5948 protein coding "SMMC-7721,HepG2" -- Hepatocellular carcinoma Prevent Knockdown//SA-β-gal activity assay Cell senescence was induced with RBP2 siRNA knockdown for 72 hr in cancer cells. CDKIsp16//p21CIP2//p27//hsa-miR-212 --//--//--//-- Knockdown//Western blot//CHIP//Luciferase reporter assay//qRT-PCR "The expression of known targets of RBP2, namely, the CDKIs p16ink4a, p21CIP2 and p27kip1, was markedly increased with RBP2 siRNA knockdown.siRNA knockdown of RBP2 expression greatly increased luciferase activity of p21CIP2 and slightly but significantly increased activity of p27kip1 in HepG-2 cells; ChIP assay confirmed the results.with RBP2 overexpression, the expression of p21CIP2 and p27kip1 was relatively low .RBP2 expression was markedly repressed with hsa-miR-212 overexpression. To further confirm that RBP2 was negatively regulated by hsa-miR-212, transfection of hsa-miR-212 inhibitor plasmid in the 2 cell lines decreased the expression of hsa-miR-212 and increased that of RBP2." -- -- -- -- Human HL cellular senescence 23922798 Sen_G_0195 NOX1 27035 protein coding "REF52,TIG-3" -- Aging Accelerate SA-β-gal activity assay "Nox1- and Nox4- overexpressing cells ceased to proliferate and accumulated SA-β-gal during 6–7?days postinfection, whereas vector control displayed little or no senescence biomarker and growth inhibition." -- -- -- -- p19//p38 MAPK Upregulation//Upregulation Western blot "Nox1 up‐regulated the expression of p19Arf?in REF52 cells.Moreover, a marked increase in the level of phosphorylated p38MAPK was detected in Nox1‐ or Nox4‐expressing cells ." Human L cellular senescence 23216904 Sen_G_0196 NOX4 50507 protein coding "REF52,TIG-3" -- Aging Accelerate SA-β-gal activity assay "Nox1- and Nox4-overexpressing cells ceased to proliferate and accumulated SA‐β‐gal during 6–7?days postinfection, whereas vector control displayed little or no senescence biomarker and growth inhibition." -- -- -- -- p16//p38 MAPK Upregulation//Upregulation Western blot " Nox4 caused accumulation of p16Ink4a?in TIG‐3 cells.Moreover, a marked increase in the level of phosphorylated p38MAPK was detected in Nox1‐ or Nox4‐expressing cells." Human L cellular senescence 23216904 Sen_G_0197 HRAS 3265 protein coding "TIG-3,REF52" -- Aging Accelerate SA-β-gal activity assay//Western blot "H‐RasV12‐infected cells displayed a flat, enlarged morphology and had arrested growth by 7?days after infection, which was accompanied by an increase in the activity of a senescence-associated β-galactosidase.?Furthermore, oncogenic Ras elevated the expression of senescence‐related cell cycle regulatory proteins: p53, p21 and p19Arf in REF52 cells and p53, p21 and p16Ink4a in TIG-3 cells." NOX1//NOX4 --//-- Knockdown//Western blot//SA-β-gal activity assay "Nox1 siRNA and Nox4 siRNA consistently abrogated both the accumulation of SA‐β‐gal and up‐regulation of p53, p21 or p16Ink4a in H‐RasV12‐transduced cells." p53-p21//p16 Upregulation//Upregulation Knockdown//Western blot//SA-β-gal activity assay "Nox1 siRNA and Nox4 siRNA consistently abrogated both the accumulation of SA‐β‐gal and up‐regulation of p53, p21 or p16Ink4a in H‐RasV12‐transduced cells." Human L cellular senescence 23216904 Sen_G_0198 AKT1 207 protein coding U87 -- Glioma Prevent SA-β-gal activity assay The cytoprotective effects of Akt overexpression were associated with a reduction in the percentage of cells expressing senescence-associated β-galactosidase activity at 10 days following temozolomide exposure. Chk2 -- Western blot "U87MG cells expressing the AktERM+ construct, however, exhibited a statistically significant reduction in temozolomide-induced Chk2 phosphorylation (but not in total Chk2 levels) relative to that noted in cells not overexpressing Akt." -- -- -- -- Human L cellular senescence 15930307 Sen_G_0199 RB1 5925 protein coding "H1299,H460" -- Lung cancer Accelerate SA-β-gal activity assay "RB-proficient and RB-deficient H1299 and H460 cells were exposed to PD 0332991 for two weeks and analyzed for the expression of the senescence marker β-galactosidase activity. Interestingly, elevated levels of β-galactosidase activity were observed in RB-proficient cells in response to PD 0332991, while RB-deficient cells failed to induce β-galactosidase, suggesting an RB-dependent senescence program." -- -- -- -- -- -- -- -- Human HL cellular senescence 29311118 Sen_G_0200 MAP2K4 6416 protein coding MEF -- Aging Prevent SA-β-gal activity assay//Knockdown "As expected, MKK4-KO MEFs, in which the non-canonical NFκB pathway is inactivated, showed more prominent β-galactosidase activity than MKK4-WT when tested in a senescence-associated β-gal activity assay, indicating that MKK4 depletion increased cellular senescence." p16//p21//p27 --//--//-- Western blot "MKK4 depletion enhanced the protein levels of cyclin-dependent kinase (CDK) inhibitors, p16, p21, and p27, which in turn induced cell growth retardation." NF-κB Activation Western blot "To investigate the direct roles of the non-canonical NFκB pathway, we knocked down NFκB2 using shRNAs in MKK4-WT MEFs.As with MKK4 depletion, NFκB2 knock-down also suppressed cell growth." Human L cellular senescence 28733031 Sen_G_0201 CIB1 10519 protein coding MCF-7 -- Aging Prevent Knockdown//SA-β-gal activity assay "About 65% of KIP knockdown cells were stained intensely for senescence associated β-galactosidase (SA-β-Gal) activity and exhibited a senescent phenotype at day 15.In contrast, the growth rates of two independent KIP knockdown cells gradually slowed down and almost stopped dividing at ~5 PD." TRF2 Binding Pull-down assay "To verify the direct interaction between TRF2 and KIP , we performed GST pulldown experiments. GST-KIP , but not the control GST, bound to FLAG-TRF2 expressed in MCF7 cells, indicating that KIP interacts with TRF2 in vitro." -- -- -- -- Human HL cellular senescence 25012820 Sen_G_0202 PPIB 5479 protein coding U251 -- Glioblastoma Prevent Knockdown//SA-β-gal activity assay "As suggested by these findings, although the majority of U251 cells died upon CypB knockdown, surviving cells showed microscopic changes characteristic of senescence, including large and flat morphology, binuclear cells, and massive vacuolization. Furthermore, CypB knockdown in U87 cells markedly increased SA-β-gal staining.Overexpression of oncogenic Ras (HRASV12) in GBM cells evoked similar senescence-associated morphological and molecular changes." p27 -- Knockdown//Western blot CypB knockdown or inhibition also increased levels of the cell cycle inhibitor p27/Kip1 (24) and strongly reduced the p27-specific E3 ubiquitin ligase Skp2. -- -- -- -- Human HL cellular senescence 24272483 Sen_G_0203 NTN4 59277 protein coding U251MG -- Glioblastoma Prevent SA-β-gal activity assay We then detected the number of senescent cells by using senescence-associated beta-galactosidase assay. We observed that silencing of either NTN4 or ITGB4 significantly induced U251MG cell senescence. Next we explored the effects of exogenous recombinant NTN4 on TMZ induced senescence. NTN4 significantly inhibited U251MG cell senescence induced by TMZ. ITGB4 -- SA-β-gal activity assay "Although NTN4 reduced the senescence rate of wild type or non-targeting control U251MG cells induced by TMZ treatment,NTN4 did not affect ITGB4-silenced U251MG cells.This result indicates that ITGB4 mediates the protective effect of NTN4 on TMZ treated glioblastoma cells." -- -- -- -- Human L cellular senescence 24265816 Sen_G_0204 ITGB4 3691 protein coding "U251MG,U-118 MG,T98G,U87 MG" -- Glioblastoma Prevent SA-β-gal activity assay//Knockdown "We observed that silencing of either NTN4 or ITGB4 significantly induced U251MG cell senescence.Similar results were obtained by silencing the expression of either NTN4 or ITGB4 in U118MG, T98G and U87MG cells." -- -- -- -- -- -- -- -- Human L cellular senescence 24265816 Sen_G_0205 SORBS2 8470 protein coding Primary human keratinocyte -- Aging Accelerate SA-β-gal activity assay "The highest number of senescent cells was observed for SORBS2-2 fourteen days after transduction.The characteristic perinuclear β-galactosidase staining pattern of enlarged and flattened cells was observed in case of fibroblasts.In contrast, the morphology of senescing keratinocytes differed in that both enlarged and rounded cells with an overall blue stain were observed. SORBS2-2 and TLR3 could induce senescence in both cell types." -- -- -- -- -- -- -- -- Human HL cellular senescence 24165198 Sen_G_0206 TLR3 7098 protein coding Primary human Fibroblast -- Aging Accelerate SA-β-gal activity assay "The highest number of senescent cells was observed for SORBS2-2 fourteen days after transduction.The characteristic perinuclear β-galactosidase staining pattern of enlarged and flattened cells was observed in case of fibroblasts.In contrast, the morphology of senescing keratinocytes differed in that both enlarged and rounded cells with an overall blue stain were observed. SORBS2-2 and TLR3 could induce senescence in both cell types." -- -- -- -- -- -- -- -- Human HL cellular senescence 24165198 Sen_G_0207 HDAC7 51564 protein coding "HeLa,MCF-7" -- Cancer Prevent Knockdown//Flow cytometry//SA-β-gal activity assay//BrdU assay "HDAC7 knockdown by all the three pairs of oligos significantly decreased the proportion of cells in S phase and increased G0/G1 cell proportion, indicating a G1/S cell cycle arrest. Congruent with the above findings, HDAC7-silenced cells revealed a significant loss of BrdU labeling (16.9%) compared with the control cells (33.2% of BrdU incorporation), suggesting a reduced entry into S phase.HDAC7 silencing markedly elevated the SA-β-Gal activity in HeLa cells (from 0.9% to 16.8%) and MCF-7 cells (from 7.8% to 27.1%)." c-Myc//p21//p27 --//--//-- Western blot "Immunoblot analysis showed that the expression level of c-Myc was remarkably suppressed by HDAC7 silencing, while p21 and p27 levels were drastically upregulated." -- -- -- -- Human L cellular senescence 21120446 Sen_G_0208 IRF5 3663 protein coding MDAH087-1 -- Aging Accelerate SA-β-gal activity assay "IRF5 or IRF7, induced an additional 5% senescence-associated β-gal staining over background. The combination of IRF5 with IRF7 was able to induce senescence-associated β-gal activity in 19.3% of the cells, which is equivalent to that observed in 5-aza-dC-treated immortal MDAH041 cultures. Similar results were observed in IRF5 + IRF7-overexpressing MDAH087-1 cells." -- -- -- -- -- -- -- -- Human HL cellular senescence 18505922 Sen_G_0209 IRF7 3665 protein coding MDAH087-1 -- Aging Accelerate SA-β-gal activity assay "IRF5 or IRF7, induced an additional 5% senescence-associated β-gal staining over background. The combination of IRF5 with IRF7 was able to induce senescence-associated β-gal activity in 19.3% of the cells, which is equivalent to that observed in 5-aza-dC-treated immortal MDAH041 cultures. Similar results were observed in IRF5 + IRF7-overexpressing MDAH087-1 cells." -- -- -- -- -- -- -- -- Human HL cellular senescence 18505922 Sen_G_0210 DEK 7913 protein coding HeLa -- Aging Prevent SA-β-gal activity assay "Whereas all of the AdE2ts-infected control cells incubated at the permissive temperature exhibited the characteristically senescent morphology, DEK expression resulted in a mixture of cells with senescent as well as nonsenescent features, indicating partial repression of the E2 senescence phenotype.In agreement with these morphological findings, over 70% of the control cell population were positive for the senescence-specific SA–β-Gal marker at 32°C, with a reduction to 15% upon DEK overexpression." -- -- -- -- -- -- -- -- Human HL cellular senescence 16254365 Sen_G_0211 ACACA 31 protein coding Human primary Fibroblast -- Aging Prevent BrdU assay//SA-β-gal activity assay "The incorporation of [methyl-3H] thymidine into DNA, normalized to total cell protein, was significantly reduced,as was the percentage of cell nuclei that incorporated BrdU. Besides, a nearly three-fold increase in SA-β-Gal positive cells was observed after silencing of ACC1 gene expression." p38MAPK -- Western blot "p38 MAPK phosphorylation was stimulated by ACC1 silencing, while the overall expression of the kinase was not affected . As such, the ratio between the phosphorylated enzyme and total enzyme was more than three-fold higher in ACC1 shRNA treated cells than in the control." -- -- -- -- Human L cellular senescence 27983949 Sen_G_0212 S100A6 6277 protein coding HUVEC -- Aging Prevent SA-β-gal activity assay//Knockdown "Compared with control cells treated with scrambled siRNA,S100A6-depleted cells appeared larger and more ""fluffy"". Analysis showed that these larger, ""fluffy"" HUVECs exhibited blue staining characteristic of β-galactosidase expression, with a higher incidence of blue cells occurring upon depletion of S100A6. Quantification revealed an increase in β-galactosidase levels of greater than four-fold upon S100A6 depletion." CDK1 -- Western blot "Depletion of endothelial S100A6 caused a decrease in both CDK1 and phospho-CDK1 levels in subconfluent HUVECs,Importantly, in HUVECs, depletion of the S100A6 levels by S100A6 siRNA#3 caused a decrease of almost 50% in the levels of CDK1 mRNA." -- -- -- -- Human HL cellular senescence 23095053 Sen_G_0213 CARM1 10498 protein coding Human bronchial epithelial cell -- Chronic obstructive pulmonary disease Prevent SA-β-gal activity assay//Knockdown "There was a significant increase in the number of beta-galactosidase positive cells in the airways of NA-treated Carm1+/- mice compared to NA-treated WT animals (31.8 ± 3.4% vs 8.2 ± 2.8%, p<0.001). Consistent with mouse airway epithelial cells, the siCARM1-transfected 337 human cells showed a significantly higher percentage of beta-galact osidase-positive cells compared with 338 scrambled controls (13.2 ± 0.6% vs 3.7 ± 0.6%, p<0.001)." SIRT1//p53 --//-- Western blot//qRT-PCR "Moreover, western blot analysis of whole lung homogenates from d14 mice treated with NA revealed a clear reduction in the level of SIRT1 protein in Carm1+/- mice compared to WT controls, with a concomitant increase in p53 levels .Mechanistically, CARM1-silenced cells demonstrated reduced expression of SIRT1, and treating CARM1-silenced cells with the SIRT1 activator resveratrol, reversed the impaired wound healing." -- -- -- -- Human HL cellular senescence 31461302 Sen_G_0214 KDM3A 55818 protein coding "OVCAR-5,A2780" -- Ovarian cancer Prevent SA-β-gal activity assay//Knockdown//Flow cytometry "Interestingly,KDM3A depletion led to G2/M cell cycle arrest, which was accompanied by increased apoptosis,Since we observed that growth arrest was accompanied by the presence of large, flat and vacuolated cells in KDM3A knockdown cells,Indeed,both OVCAR-5/CDDP and A2780/CDDP cells expressing KDM3A shRNAs showed high abundance of senescence-associated β-galactosidase positive cells as compared with scrambled control cells,indicating that KDM3A prevents the cellular senescence to promote sustained growth of ovarian cancer." -- -- -- -- -- -- -- -- Human HL cellular senescence 27694900 Sen_G_0215 CLU 1191 protein coding IMR-90 -- Aging Prevent SA-β-gal activity assay//Knockdown "Enhanced senescence was confirmed by significant differences in the SA-β-gal+ cells, where at least two-fold increases were noted compared to untreated pTRIPZ-shCLU IMR-90 cells that expressed sCLU at day 39. Dox-inducible non-silencing shRNA had no affect on sCLU expression, both population doubling times and %SA-β-gal+ cells were not significantly different with or without Dox exposure over the life of the IMR-90 cultures." ATM -- Western blot AT2052 cells expressed significantly lower basal level expression of sCLU compared to wild-type ATM+ IMR-90 cells. IGF-1R-MAPK-ERK1/2-Egr-1 -- ELISA//Western blot//TUNEL assay "Indeed, IGF-1 expression was approximately two-fold higher in conditioned media from senescent compared to young IMR-90 cells. Analyses of differential signal transduction responses in senescent versus young IMR-90 cells showed that IGF-1 receptor (IGF-1R) levels were activated (elevated P-IGF-1R/tIGF-1R) in senescent cells; antibodies to human IGF-1R are not ideal. Src and Erk-1/2 kinase levels were also elevated in middleaged and senescent compared to young IMR-90 cells. The transcription factor, Egr-1, which binds the hCLU promoter and mediates sCLU expression, was significantly increased during senescence in IMR-90 cells. To further confirm the specificity of IGF-1/IGF-1R/MAPK/ERK-1/2/Egr-1 signaling in sCLU induction during senescence, we treated senescent IMR90 cells with the IGF-1R inhibitor, AG1024, which effectively blocked IR-induced sCLU expression and also suppressed sCLU expression in senescent IMR-90 cells in a dose-dependent manner ." Human L cellular senescence 24937130 Sen_G_0216 ING1 3621 protein coding Fibroblast -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis "Retroviral transduction with p33ING1 resulted in a change to senescence-like cell morphology, as documented by phase-contrast microscopy.We tested for expression of SA-β-Gal activity in p33ING1-infected primary human fibroblasts by use of RasV12 as a control. In accordance with the previous observations, p33ING1 induces SA-β-Gal activity to a extent similar to that seen with oncogenic Ras, suggesting that p33ING1 has potency similar to that of RasV12." -- -- -- -- -- -- -- -- Human L cellular senescence 15601862 Sen_G_0217 SFRP1 6422 protein coding CSC -- Aging Accelerate SA-β-gal activity assay//Western blot//ELISA "We also confirmed the up-regulation of senescence markers p16, p21, phosphorylated p53, and SA-β-gal in sFRP1-treated CDCs.Importantly, sFRP1-treated CDCs presented SASP, which was characterized by up-regulation of IL-6 and IL-1b, and was consistent with anti-cancer drug-induced cellular senescence." -- -- -- -- Wnt Downregulation Western blot "Different concentrations of recombinant human sFRP1 were administered to CDC cultures for 1 and 48 h. Recombinant sFRP1 treatment caused an increase in phosphorylated b-catenin and a decrease in phosphorylated GSK3b in young patients-derived CDCs,indicating that exogenous sFRP1 inhibited canonical Wnt signaling." Human L cellular senescence 28435069 Sen_G_0218 TP53 7157 protein coding PCI-13 -- Squamous cell carcinoma Accelerate Colony formation assay//SA-β-gal activity assay "Clonogenic assays showed that the 4-Gy surviving fraction in TP53 null or mutant cells was significantly higher than in cells with wtp53. Being that senescence is the primary mechanism of radiation-induced cell death in HNSCC cells, a senescence-associated β-galactosidase (SA-β-gal) staining was performed and showed that 4 days after treatment with 4-Gy ionizing radiation, cells with wtp53 had a significantly higher SA-β-gal-positive staining compared with the SA-β-galpositive staining in TP53 null and all TP53 mutant cells." p21 Upregulation qRT-PCR//Western blot "Among the mutp53 bearing cells, only PCI-13 A161S had minimal, short-lived expression of the p21 gene compared with the enhanced, long-term expression seen in wtp53 cells. All other mutp53 or TP53 null cell lines had no significant elevation in p21 mRNA. Western blot analysis demonstrated long-term expression of the p21 protein in wtp53 cells, with no expression in mutp53 and TP53 null cells, but short-term expression in PCI-13 A161S cells." -- -- -- -- Human L cellular senescence 25766317 Sen_G_0219 NDRG1 10397 protein coding HepG2 -- Hepatocellular carcinoma Prevent SA-β-gal activity assay//Knockdown A large percentage (about 80%) of HepG2 cells transfected with NDRG1-specific siRNA was positive for SA-β-Gal staining. -- -- -- -- GSK3β-p53/-p16 -- SA-β-gal activity assay//Western blot "The percentage of SA-β-Gal positive cells was also reduced in HepG2 cells co-transfected with NDRG1-specific siRNA and siRNA against p53, p16, p53 & p16, or GSK-3. Western blot results indicated that co-transfection of NDRG1-specific siRNA and siRNA against p53, p16, or p53 & p16 increased p-Rb level which was decreased by NDRG1-specific siRNA . Additionally, GSK-3β siRNA decreased GSK-3β, GSK-3β 9ser, p53, p21 and p16 levels, indicating that GSK-3β can stabilize p53 and p16 levels in HepG2 cells with NDRG1 suppression. Consistently, a decrease in p21 (a p53 downstream target) and increased p-Rb were detected in cells co-treated with NDRG1-siRNA and PFT-α." Human HL cellular senescence 24302615 Sen_G_0220 RB1 5925 protein coding MSC -- Aging Prevent SA-β-gal activity assay//Cell morphological analysis//Knockdown "We observed signs of senescence in shR1 as detected by in situ senescence-associated-beta-galactosidase assay, compared with cells transduced with controls shRNAs.Beta-galactosidasepositive cells showed the characteristic senescent morphology (flat, enlarged cells). Moreover, we detected an increase in the percentage of bi-nucleate cells in shR1 (data not shown), which is another hallmark of senescent cells." p53//p27//p21 --//--//-- RT-PCR//Western blot "We detected a significant up-regulation (p < 0.05) of p53 protein in shR1 along with an increase in the level of p27kip1 and p21cip1, whereas no modification was observed in p16Ink4a. the increased expression of these genes was also observed at mRNA level." -- -- -- -- Human HL cellular senescence 23370776 Sen_G_0221 KIR2DL4 3805 protein coding NK -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis//Flow cytometry "Primary NK cells had a characteristicflattened and enlarged morphology after activation through CD158d. The average 2D cell area of NK cells activated by CD158d was 19.5 ±11 μm2(n = 23), up from 6.6 ±2.4 μm2(n = 22)for cells stimulated with control Ab.There was an increase in senescence-associated β-galactosidase (SA-β-gal) activity, a widely used senescence biomarker.Furthermore, NK cells activated by CD158d were arrested at the G0/G1 stage of the cell cycle." -- -- -- -- -- -- -- -- Human HL cellular senescence 23184984 Sen_G_0222 NOX4 50507 protein coding MCF12A -- Breast cancer Accelerate SA-β-gal activity assay "We performed a SA beta-galactosidase assay on the empty vector and NOX4 overexpressing cells, and determined a higher instance of senescence in the NOX4 overexpressing cell lines when compared to the empty vector controls. An increase in senescence was observed in both breast epithelial and cancer cell lines when NOX4 was overexpressed." -- -- -- -- -- -- -- -- Human HL cellular senescence 20523116 Sen_G_0223 TNF 7124 protein coding EPC -- Aging Accelerate SA-β-gal activity assay Continuous treatment with TNF-α for 1 wk resulted in a dose-dependent increase in EPC senescence. There was a dose-dependent drop in the EPC population doubling rate with TNF-α treatment that mirrored the observed increase in cell senescence measured by SA-β-Gal staining. p16 Upregulation Flow cytometry "We observed a marked up-regulation of p16INK4a expression after the continuous TNF-α treatment, as is shown in a representative flow cytometry histogram. " p38 MAPK Activation Western blot "We first demonstrated that the p38 MAPK pathway was activated by TNF-α stimulation, as evidenced by phosphorylation of p38, and that the p38 MAPK inhibitor SB203580 is able to block this activation at a concentration of 10uM." Human L cellular senescence 19124561 Sen_G_0224 MDM2 4193 protein coding Adipose stromal cell -- Aging Prevent SA-β-gal activity assay//qRT-PCR//Cell morphological analysis "Furthermore, as expected, antagonizing MDM2 decreased the expression of HMGA2 significantly supporting the assumption that the p14Arfrepression of HMGA2 is TP53-dependent.Thus, antagonizing MDM2 can induce senescence in mesenchyme-derived stem cells. The reduced levels of HMGA2 expression are also accompanied by an increased number of β-gal positive cells. For these experiments, canine ADSCs were treated with 30 lM and 50 lM nutlin-3, respectively, for 72 hr. The number of β-gal positive cells was then determined by counting 500–600 cells. With both concentrations,the percentage of positive cells was drastically increased (30 lM: 54.0%; 50 lM: 60.0%) compared with the control cells (9.9%). Also, the increased percentage of positive cells was accompanied by clear morphological changes leading to a majority of flat enlarged cells with an absence of mitoses." HMGA2 -- qRT-PCR "Furthermore, as expected, antagonizing MDM2 decreased the expression of HMGA2 significantly." -- -- -- -- Human L cellular senescence 21456046 Sen_G_0225 CCL2 6347 protein coding UCB-MSC -- Aging Accelerate SA-β-gal activity assay//Knockdown "Compared with the untreated control, MCP-1 treatments significantly increased SA-β-gal activity with passaging by up to 8-fold, concomitantly with a considerable delay in cell growth.The use of siRNA to silence MCP-1 in UCB-MSCs significantly blocked both SA-β-gal activity induction and delayed cell growth ." CCR2 -- "Flow cytometry//Western blot//SA-β-gal activity assay//Cell growth kinetics//Colony formation assay " "MCP-1 is a pro-inflammatory cytokine that exerts its biological effects through its cognate receptor CCR2 (5,45). The expression of CCR2 in UCB-MSCs was lower in the early-phase but it was considerably upregulated in the intermediate-phase and further increased in the terminal senescent phase. Importantly, treatment with CCR2 blocking antibody significantly reduced SA-β-gal expression and enhanced the growth rate and colony-forming capacity of UCB-MSCs in the intermediate-phase." ROS-p38-MAPK-p53-p21 Activation Western blot//Knockdown "MCP-1 treatment activated the p53-p21 signaling cascade, increasing the protein level of p53 phosphorylated at Ser392 and total p21.At the molecular level, MCP-1 KD cells showed attenuated activation of the p53-p21 pathway.Moreover, p38-MAPK, a crucial mediator of ROS-induced senescence, was activated from 8 h and peaked at 18 h after MCP-1 stimulation .Importantly, treatment with SB203580, a chemical inhibitor of p38-MAPK, significantly abrogated the increase in phosphorylated p53 and p21 proteins induced by MCP-1 stimulation." Human HL cellular senescence 26573462 Sen_G_0226 CCR2 729230 protein coding UCB-MSC -- Aging Accelerate Western blot//Knockdown//SA-β-gal activity assay "The significance of CCR2 was further validated by the fact that CCR2 KD cells had decreased SA-β-gal activity and p53-p21 pathway activation. Furthermore, overexpression of CCR2 in early-phase UCB-MSCs remarkably increased cellular senescence, as indicated by induction of SA-β-gal activity , activation of the p53-p21 signaling cascade, and downregulation of BMI1 expression." BMI1 Downregulation Western blot//Knockdown//SA-β-gal activity assay "The significance of CCR2 was further validated by the fact that CCR2 KD cells had decreased SA-β-gal activity and p53-p21 pathway activation. Furthermore, overexpression of CCR2 in early-phase UCB-MSCs remarkably increased cellular senescence, as indicated by induction of SA-β-gal activity, activation of the p53-p21 signaling cascade, and downregulation of BMI1 expression." p53-p21 Activation Western blot//Knockdown//SA-β-gal activity assay "The significance of CCR2 was further validated by the fact that CCR2 KD cells had decreased SA-β-gal activity and p53-p21 pathway activation . Furthermore, overexpression of CCR2 in early-phase UCB-MSCs remarkably increased cellular senescence, as indicated by induction of SA-β-gal activity, activation of the p53-p21 signaling cascade, and downregulation of BMI1 expression." Human HL cellular senescence 26573462 Sen_G_0227 BMI1 648 protein coding UCB-MSC -- Aging Prevent SA-β-gal activity assay//Knockdown "As previously reported, BMI1 KD UCB-MSCs exhibited the typical senescent phenotype, such as an enlarged and flattened morphology and increased SA-β-gal activity." MCP-1 -- Western blot//Knockdown//SA-β-gal activity assay "As expected, the promoters of MCP-1 in BMI1 KD cells lost the recruitment of BMI1 and the concomitant H2AK119Ub histone modification . The epigenetic change in the MCP-1 locus in BMI1 KD cells coincided with derepression of MCP-1 transcription, which resulted in increased secretion of MCP-1 protein. The enhanced secretion of MCP-1 protein in BMI1 KD cells triggered cellular senescence, as judged by the increase in SA-β-gal activity and the activation of the p53-p21 signaling pathway. Most importantly, the senescent phenotypes shown in BMI1 KD cells were significantly rescued after silencing of MCP-1." p53-p21 -- Western blot//Knockdown//SA-β-gal activity assay "The enhanced secretion of MCP-1 protein in BMI1 KD cells triggered cellular senescence, as judged by the increase in SA-β-gal activity and the activation of the p53-p21 signaling pathway ." Human HL cellular senescence 26573462 Sen_G_0228 RBP2 5948 protein coding "AGS,HeLa,SiHa,HGC-27,BGC-823" -- Gastric cancer Prevent SA-β-gal activity assay//BrdU assay "Significant fractions of the cells treated with RBP2 siRNA became positive for SA-β-Gal staining. Consistent with positive SA-β-Gal staining, these RBP2-depleted cells exhibited significantly lower BrdU labeling than did control cells." p16//p21//p27 --//--//-- Western blot//qRT-PCR//SA-β-gal activity assay "p16ink4a, p21CIP1, and p27kip1 promoter activity significantly increased following RBP2 depletion in AGS and BGC cells. In contrast, cotransfection with the RBP2 expression vector led to a substantially declined activity of all 3 promoters." -- -- -- -- Human L cellular senescence 19850045 Sen_G_0229 PIM1 5292 protein coding 2BS -- Aging Accelerate Western blot//Knockdown//SA-β-gal activity assay//MTT assay "Additionally, Pim-1 overexpression increased levels of the senescence-associated protein p16 and decreased levels of the DNA methyltransferase DNMT1, which is known to negatively correlate with senescence.Conversely, HBP1 knockdown attenuated the effect of Pim-1 on the expression of these two genes. HBP1 knockdown also attenuated Pim-1-induced senescence as indicated by SA-β-gal staining and growth arrest in 2BS cells, suggesting that HBP1 also participates in Pim-1-induced premature senescence in normal cells." HBP1 Binding Western blot "Exogenous Pim-1 increased HBP1 protein levels but had no effect on HBP1 mRNA expression. Moreover, Pim-1 knockdown decreased HBP1 protein levels but had no effect on HBP1 mRNA." -- -- -- -- Human HL cellular senescence 28348080 Sen_G_0230 SIRT1 23411 protein coding PAEC -- Atherosclerosis Prevent SA-β-gal activity assay//Crystal violet assay "Compared to cells transfected with pcDNA vector, the number of senescent cells was reduced significantly in PAECs overexpressing hSIRT1 (by ~42%) or S47A (by ~56%), and the proliferation was enhanced significantly by augmenting either of these two proteins." CDK5 Binding Co-IP//Western blot "Direct protein-protein interaction between SIRT1 and endogenous CDK5 was confirmed by co-immunoprecipitation. Using the M2 anti-FLAG agarose beads, CDK5 was detected in the immune-complexes precipitated from PAECs transiently transfected with Flag-hSIRT1-WT but not pcDNA vector. The in vitro phosphorylation assay further validated the ability of CDK5 complexes to phosphorylate SIRT1 at S47." -- -- -- -- Human HL cellular senescence 22753194 Sen_G_0231 OTX2 5015 protein coding "Daoy ,MED8A" -- Medulloblastoma Accelerate SA-β-gal activity assay//Cell morphological analysis "Furthermore,sustained induction of OTX2 in MED8A or DAOY cells resulted in clear morphologic changes. Cells became flattened and showed an increase in the amount of cytoplasm .β-Galactosidase activity was detected in cells withOTX2expression, but not in control cells." IL-6//IGFBP7 Upregulation//Upregulation Western blot "Indeed,the expression of senescence-associated secretory factors IL-6 (27) and IGFBP7 (28) was strongly increased after OTX2 induction ." p53 Activation Luciferase reporter assay "To confirm P53 activation after OTX2 induction, we transfected wild-type and mutant reporter constructs for P53 activity into MED8A-OTX2 and MED8A-control cells. The results plotted show an up to 4-fold increase in luciferase activity after OTX2 induction in MED8A-OTX2 cells, but not in MED8A-control cells or in MED8A-OTX2 cells transfected with a mutated reporter construct." Human HL cellular senescence 21047732 Sen_G_0232 ZEB1 6935 protein coding EPC1-hTERT -- Endometrial cancer Prevent SA-β-gal activity assay//Western blot "Stable knockdown of either ZEB1 or ZEB2 resulted in upregulation of p15INK4B and p16INK4A, accompanied by transcriptional activation of the respective promoters, and senescence in a subset of the cells, indicating the possibility that ZEB may mitigate EGFR-induced senescence." p15INK4b//p16 Upregulation//Upregulation Western blot "ZEB1 or ZEB2 resulted in upregulation of p15INK4B and p16INK4A, accompanied by transcriptional activation of the respective promoters." -- -- -- -- Human L cellular senescence 20424117 Sen_G_0233 ZEB2 9839 protein coding EPC2-hTERT -- Endometrial cancer Prevent SA-β-gal activity assay//Western blot//Knockdown "Stable knockdown of either ZEB1 or ZEB2 resulted in upregulation of p15INK4B and p16INK4A, accompanied by transcriptional activation of the respective promoters , and senescence in a subset of the cells, indicating the possibility that ZEB may mitigate EGFR-induced senescence." p15INK4b//p16 Upregulation//Upregulation Western blot "ZEB1 or ZEB2 resulted in upregulation of p15INK4B and p16INK4A, accompanied by transcriptional activation of the respective promoters." -- -- -- -- Human L cellular senescence 20424117 Sen_G_0234 EGFR 1956 protein coding EPC2-hTERT -- Endometrial cancer Accelerate SA-β-gal activity assay//Western blot "We suspected that EGFR overexpression may trigger senescence. In fact, senescence was observed in 30–40% of EPC2-hTERT cells shortly after drug selection upon retrovirus-mediated transduction of EGFR, but not a control empty vector." -- -- -- -- -- -- -- -- Human L cellular senescence 20424117 Sen_G_0235 PPARGC1A 10891 protein coding HBMVEC -- Aging Prevent Knockdown//SA-β-gal activity assay//Tube formation assay//Cell counting//Cell migration assay "PGC1α downregulation also induced cellular senescence-associated phenotypes, such as decreased tube formation, cell proliferation, and cell migration and increased SA-β-gal activity,suggesting that PGC1α contributes to endothelial cell senescence by maintaining mitochondrial function.and the overexpression of PGC1α in old cells increased cell proliferation and partially reduced the senescent phenotype as shown by SA-β-gal staining." HDAC1//SIRT1 --//-- Knockdown//Western blot Reduction of HDAC1 by transfection of HDAC1 siRNA into cells increased PGC1α acetylation.Sirt1 knockdown also induced PGC1α acetylation. -- -- -- -- Human L cellular senescence 30016403 Sen_G_0236 CDKN2A 1029 protein coding iCSCL-10A -- Cancer Accelerate Cell cycle analysis//Cell morphological analysis//Flow cytometry//SA-β-gal activity assay "Cell cycle analysis demonstrated a significantly increased G1 population of p16INK4a-transduced cells compared with the control vector-transduced cells .Cells transduced with p16INK4aexhibited an enlarged, flattened and irregular shape, and flow cytometric analysis revealed this effect in terms of forward scatter and side scatter profiles.These cells also positively stained with senescence-associated β-galactosidase, whereas no such cells were observed among the control vector-transduced population." -- -- -- -- -- -- -- -- Human HL cellular senescence 23318426 Sen_G_0237 MCRS1 10445 protein coding "IMR-90,Hs68" -- Aging Accelerate PI staining//SA-β-gal activity assay//Cell morphological analysis "To confirm that MSP58-overexpressing cells entered senescence, we analyzed DNA content using flow cytometry with propidium iodide staining. HT1080 MSP58 clone 11 and 14 cells showed decreased fractions in the G1phase resulting from cells accumulating in the S and G2/M phases and elevated forward and side scatter relative to control cells, respectively reflecting increased cell size and granularity.Notably, stable HT1080 and NIH3T3 transfectants expressing MSP58 showed elevated SA-β-gal activity, a classical biomarker of senescence.After selection, MSP58-infected cells displayed a large size with a flattened shape and significantly increased SA-β-gal activity compared with control cells." BRG1 Binding Yeast Two-Hybrid assay//Co-IP//Western blot MSP58 selectively bound to the N-terminal domain (amino acids 1 750) of BRG1.Overexpressed MSP58 and BRG1 proteins were reciprocally coimmunoprecipitated in COS-1 cells. p53-p21 Activation Luciferase reporter assay//Western blot//SA-β-gal activity assay "Elevated levels of p53 and p21 proteins were also detected in MSP58-overexpressing IMR90, Hs68, and H184B5F5/M10 cells;Cells transfected with the truncated constructs showed substantially reduced responses as the p21-dm construct was not induced, indicating that MSP58 regulates p21 promoter activity through the p53 protein.Furthermore, depletion of ATM or p21 using shRNA transfection significantly reduced the incidence of MSP58-induced senescent phenotypes as shown by the representative stable clones HT1080 MSP58 clone 11-ATMsi 9 and MSP58 clone 11-p21si6." Human L cellular senescence 22563078 Sen_G_0238 EID3 493861 protein coding MCF-7 -- Breast cancer Accelerate SA-β-gal activity assay "Induction of EID3 expression with doxycycline augmented the effects of IR on the cells, resulting in a significant increase in the induction of SA-β-gal positive senescent cells compared to the cells exposed to IR alone without the induction of EID3 by doxycycline." p21//p57 Upregulation//Upregulation qRT-PCR//Western blot The increase in p21 and p57 expression was augmented by the induction of EID3 with doxycycline. -- -- -- -- Human HL cellular senescence 30114644 Sen_G_0239 CHD5 26038 protein coding "HepG2,Hep3B" -- Hepatocellular carcinoma Accelerate WST-8 assay//SA-β-gal activity assay "Similarly, the proliferation of Hep3B-CHD5 was lower than Hep3B-empty vector. Consistent with the lack of proliferative capacity, HepG2-CHD5 and Hep3B-CHD5 expressed senescence-associated β-galac-tosidase (SA-β-gal) activity, a marker of senescent cells. However, very few senescent cells were detected in HepG2-empty vector and Hep3B-empty vector cells." -- -- -- -- -- -- -- -- Human HL cellular senescence 24529164 Sen_G_0240 BCL11B 64919 protein coding "U87,U251" -- Glioma Prevent Knockdown//SA-β-gal activity assay//Cell morphological analysis//PI staining "The histochemical assay for SA-β-Gal activity showed that SA-β-Gal activity was strongly produced in U87-KD1 and U87-KD2 cells with a flat, enlarged cell shape . The extensive SA-β-Gal activity was also detected in U251-KD1 and U251-KD2 cells compared to that of U251-Mock cells. We noted that the activity of SA-β-Gal was weak in either U87- or U251-Mock cells.The percentage of U87-KD1 and U87-KD2 cells at the G0/G1 phase was increased when compared to that observed in U87-Mock cells. In addition, the reduction of Bcl11b expression caused an increase in the arrest of U251 cells at the G0/G1 (KD1) or S (KD2) phase, along with a reduction in the amount of cells at the G2/M phase." p21//p53//SOX2//Bmi-1 --//--//--//-- qPCR//Western blot "Similar to p21, the production of p53 mRNA and proteins was increased in C6 and U87 cells after Bcl11b gene knockdown.In addition, the transcripts of the stemness genes, Sox-2 and Bmi-1, were downregulated in U87 and U251 after Bcl11b knockdown, as well as their protein levels.Similar to p21, the production of p53 mRNA and proteins was increased in C6 and U87 cells after Bcl11b gene knockdown." -- -- -- -- Human L cellular senescence 26096706 Sen_G_0241 SIRT6 51548 protein coding NHAC-kn -- Aging Prevent SA-β-gal activity assay//Knockdown "Furthermore, the SA-β-Gal assay showed that the percentage of SA-β-Gal-positive cells was significantly higher in the SIRT6 siRNA group (24.3 ± 4.2 %) than in the control group (11.3 ± 3.0 %), indicating that the depletion of SIRT6 induced premature senescence." γH2AX//TIFs//p16//p21 --//--//--//-- Immunofluorescence microscopic analysis//Western blot Immunofluorescence microscopic analyses showed that γH2AX foci and TIFs were increased in the SIRT6-depleted chondrocytes than in controls.The p16 protein level was higher and p21 protein level was lower in the SIRT6-depleted chondrocytes than those in control chondrocytes. -- -- -- -- Human L cellular senescence 25819580 Sen_G_0242 CREB1 1385 protein coding WI-38 -- Aging Prevent SA-β-gal activity assay//Cell viability assay//Cell morphological analysis "Permanent CREB reduction in WI-38 fibroblasts highly influenced their viability and proliferation capacity.Moreover, an increased number of cells with senescence-associated β-galactosidase activity and flatten cell morphology could be observed." -- -- -- -- -- -- -- -- Human HL cellular senescence 23991634 Sen_G_0243 CDCA3 83461 protein coding "HBEC3,HBEC4,HBEC5,H2228,H460,SKMES-1,EBC-1,HTB-182,CRL-5889,A549" -- Lung cancer Prevent SA-β-gal activity assay//Cell morphological analysis NSCLC cells depleted of CDCA3 were larger than the control cells with MFI values ~4 fold greater than control cells. An enlarged cellular morphology is consistent with cells that have entered senescence.CDCA3 depletion resulted in an increased proportion of cells staining positive for SA-β-gal. p21 -- Immunofluorescence Depletion of CDCA3 induced an increase in nuclear immunofluorescent staining of p21 in all cell lines with the exception of the three HBEC cell lines. Quantification of the immunofluorescence with high content microscopy demonstrated a >3 fold increase in nuclear p21 in all NSCLC cell lines with the exception of A549 cells which demonstrated a ~2 fold increase in p21 protein. -- -- -- -- Human HL cellular senescence 28487093 Sen_G_0244 DNMT1 1786 protein coding HDF -- Aging Prevent Knockdown//SA-β-gal activity assay "Moreover, siRNA-mediated knockdown of DNMT1 alone was sufficient to induce HDF senescence." UHRF1//WNT5A --//-- Knockdown//qRT-PCR//Western blot//Flow cytometry "Although UHRF1 suppression significantly down-regulated both mRNA and protein expression of DNMT1, DNMT1 knockdown did not alter UHRF1 expression, indicating the role of UHRF1 as an upstream regulator of DNMT1 transcription. However, DNMT1 promoter activity was significantly decreased by siRNA-mediated UHRF1 suppression , further supporting regulation of DNMT1 transcriptional activity by decreased UHRF1 expression. Knockdown of either DNMT1 or UHRF1 clearly induced WNT5A protein expression, whereas HELLS knockdown showed a minor effect." -- -- -- -- Human HL cellular senescence 28100769 Sen_G_0245 UHRF1 29128 protein coding HDF -- Aging Prevent Knockdown//SA-β-gal activity assay "Compared with DNMT1 knockdown, UHRF1 knockdown more effectively led to the gain of senescent phenotypes." DNMT1//WNT5A --//-- Knockdown//qRT-PCR//Western blot//Flow cytometry "Although UHRF1 suppression significantly down-regulated both mRNA and protein expression of DNMT1, DNMT1 knockdown did not alter UHRF1 expression, indicating the role of UHRF1 as an upstream regulator of DNMT1 transcription. However, DNMT1 promoter activity was significantly decreased by siRNA-mediated UHRF1 suppression, further supporting regulation of DNMT1 transcriptional activity by decreased UHRF1 expression. Knockdown of either DNMT1 or UHRF1 clearly induced WNT5A protein expression, whereas HELLS knockdown showed a minor effect." -- -- -- -- Human HL cellular senescence 28100769 Sen_G_0246 XPG 17295032 protein coding Embryonic Fibroblast -- Aging Prevent MTT assay//Knockdown "With continuous culture, cells from -/- embryos ceased growing by 4 to 5 weeks after the start of in vitro culture, while cells from the +/+ or +/- embryos kept their growth capability 3 to 4 weeks longer, indicating that -/- cells underwent premature replication senescence,These results indicate that cells from xpg-deficient mice have a short replication life span and readily gain immortality and malignant phenotypes, suggesting that the -/- cells are genetically unstable." -- -- -- -- -- -- -- -- Human L cellular senescence 10022922 Sen_G_0247 ZNF148 7707 protein coding NCI-H460 -- Lung cancer Prevent Knockdown//SA-β-gal activity assay "Knockdown of endogenous ZBP-89 promoted NCI-H460 cell senescence, a 1.5-fold increase in SA-β-gal activity was seen after 7 days of ZBP-89i transfection, whereas overexpression of ZBP- 89 led to a reverse effect. In addition, cells transfected with ZBP-89i exhibited phenotypic changes that are typical of cells undergoing replicative senescence." p16 Downregulation SA-β-gal activity assay//Western blot//Luciferase reporter assay//RT-PCR "ZBP-89 restrained senescence of NCI-H460 cells through p16 repression. Representative photomicrographs of the SA-β-gal staining at day 7 after ZBP-89i transfection, or ZBP-89i plus p16i transfection. An irrelevant siRNA vector was used as the control.Western blotting demon- strated that p16 protein expression was decreased on ZBP-89 ectopic expression, whereas it was enhanced。Western blotting demonstrated that p16 protein expression was decreased on ZBP-89 ectopic expression, whereas it was enhanced by knockdown of the endogenous ZBP-89 in NCI- H460 cells by knockdown of the endogenous ZBP-89 in NCI- H460 cells .Overexpression of ZBP-89 greatly inhibited p16 promoter activity。The p16 mRNA level was decreased on ectopic expression of ZBP-89, but increased by knockdown of endoge- nous ZBP-89." -- -- -- -- Human L cellular senescence 19583777 Sen_G_0248 HDAC1 3065 protein coding WI-38 -- Aging Prevent SA-β-gal activity assay "Young WI-38 cells cultured in the presence of either HDAC inhibitor cells. Untreated proliferating WI-38 cells propagated in parallel had no SA-β-gal activity acquired the perinuclear staining for SA-β-gal activity normally associated with senescen，treatment with butyrate or TSA for 24 hours induced the expression of p21WAF1 in proliferating WI-38 cells. Distinct morphological changes also occurred when WI-38 cells enter replicative senescence. Senescent cells became larger and assumed irregular shapes, while proliferating WI-38 cells formed long，and striated parallel array." p21 -- Western blot Ttreatment with HDAC inhibitors butyrate or TSA induced p21WAF1 expression in proliferating WI-38 cells. -- -- -- -- Human L cellular senescence 16250917 Sen_G_0249 CD59 966 protein coding ECA-109 -- Aging Prevent Knockdown//SA-β-gal activity assay//Western blot "The results showed that significant cell cycle arrest at the G2/M phase occurred at 12, 24, and 48 h post-irradiation in CD59-deficient cells compared with that in CD59- sufficient cells .We next detected the effect of CD59 expression on the occurrence of senescence at day 6 after ionizing radiation by SA-β-gal staining. The results revealed that CD59 deficiency significantly increased the percentage of senescent Eca109 cells upon irradiation compared with that observed in CD59-sufficient Eca109 cells.The expression levels of the associated signaling molecules p21 and p16 significantly decreased at 72 h after irradiation in both CD59-sufficient and -deficient cells compared with the levels in the corresponding untreated cells, in which CD59-sufficient cells showed a greater decrease in p21 and p16 expression than CD59-deficient cells ." -- -- -- -- -- -- -- -- Human L cellular senescence 30166523 Sen_G_0250 PTTG1 9232 protein coding HCT116 -- Aging Prevent Knockdown//BrdU assay//SA-β-gal activity assay "With similar cell numbers plated, HCT116 WT and PTTG1-/- cells exhibited similar absorbance (OD 0.4) at 0 hour. After 48 hours, HCT116 WT cells showed an absorbance of 1.8 and PTTG1/- cells 1.3, suggesting that HCT116 PTTG1-/- cell number were less than WT. Reintroduction of PTTG1 into HCT116 PTTG1/- cells (PTTG1-/- +PTTG1) increased cell number after 48 hours , and these cells exhibited an OD value of 2.1 (1.6 fold increase compared to PTTG1-/- expressing an empty vector).After doxorubicin (0.005 uM) treatment, 56% of HCT116 PTTG1-/- cells exhibited enhanced β–Gal staining, compared to 5% of WT cells，we reintroduced PTTG1 into HCT116 PTTG1-/- cells，β–Gal staining indicated fewer senescent cells when PTTG1 was re-introduced (6%) than in control HCT116 PTTG1-/- cells ." p21 -- Western blot "To test the requirement of p21 for enhanced senescence observed in PTTG1-/- cells, we knocked down p21 and then assessed effects of cell treatments. PTTG1-/- HCT116 cells exhibited 21% senescent cells when p21 was suppressed, compared to 58% in control siRNA treated cells, indicating the requirement for p21 in PTTG1 mediated senescence. Moreover, HCT116 PTTG1-/- cells possess higher p21 levels." -- -- -- -- Human L cellular senescence 21858218 Sen_G_0251 GAPDH 2597 protein coding A549 -- Lung cancer Prevent Knockdown//SA-β-gal activity assay "Knockdown of GAPDH by siGAPDH caused cell proliferation arrest, in contrast to control A549 cells transfected with scrambled siRNA. Cells treated with siGAPDH manifested proliferation arrest, revealed enlarged morphology, and distinct blue staining characteristic for SA-β-galactosidase activation." AMPK//p53 --//-- Western blot//Knockdown "Following inhibition of glycolysis via GAPDH depletion,the decrease of ATP level resulted in AMPK phosphorylation, and p53 stabilization. In line with these findings, we detected accumulation of p53 phosphorylated at Ser15 in GAPDH knockdown cells." -- -- -- -- Human HL cellular senescence 21749859 Sen_G_0252 ABI3 51225 protein coding "ARO,WRO" -- Aging Accelerate Cell proliferation assay//Flow cytometry//Cell cycle analysis//SA-β-gal activity assay "Although we observed a trend toward increased apoptosis in WRO cells expressing ABI3 when compared to control cells, the growth attenuation was, however, not accompanied by a corresponding increase in apoptotic cells.The ABI3-induced growth suppression in was further studied by flow cytometric analysis, which revealed a decrease in cell viability and cell cycle arrest at the G0/G1 phase.The number of β-Gal positive cells was higher in ARO and WRO cells expressing ABI3 at days 3 and 5 postseeding." p21//E2F1 Upregulation//Downregulation RT-PCR Stable expression of ABI3 in WRO cells induced p21WAF1 while reduced E2F1 expression at days 3 and 5 post-seeding. -- -- -- -- Human L cellular senescence 21223585 Sen_G_0253 EHF 26298 protein coding MEF -- Prostate cancer Prevent Knockdown//SA-β-gal activity assay "Treatment of MEFs with the EHF-targeting siRNA resulted in decreased EHF, enlargement and flattening of the cells, and positive SA-β-Gal activity，EHF overexpression effectively rescued siEHF-induced senescence and the corresponding SA-β-Gal positivity." p16//p27 --//-- Western blot//Knockdown//SA-β-gal activity assay "EHF-knockdown cells had increased levels of p16 and p27 expression.The presence of sip27 prevented accumulation of p27 protein and reduced the SA-β-gal –positive population in MEFs and PC-3 cells, compared with controls transfected with siEHF alone." -- -- -- -- Human L cellular senescence 17172423 Sen_G_0254 LMNA 4000 protein coding "VSMC,HUVEC" -- Hutchinson-Gilford progeria syndrome Accelerate Cell morphological analysis "Introduction of progerin into VSMCs strongly reduced cell growth and shortened the replicative lifespan,In contrast, introduction of progerin had no effect on the growth and lifespan of HUVECs." p53 -- Knockdown We introduced siRNA targeting p53 into progerin-infected VSMCs and found that this siRNA counteracted the anti-proliferative effect of progerin on VSMC growth. -- -- -- -- Human HL cellular senescence 28423660 Sen_G_0255 RHOA 387 protein coding HeLa -- Aging Prevent Flow cytometry//SA-β-gal activity assay "Cell cycle analysis (by flow cytometry using PI) of irradiated cells suggested that, after exposure to 15 Gy of γ- radiation, cells with constitutively high levels of activated RhoA (HeLa-RhoA-V14) remain arrested in S and G2/M, whereas HeLa cells expressing the dominant negative RhoAN19 remain predominantly in the G1 and S phases of the cell cycle. As expected, we observed a marked arrest of HeLa cells in the G2/M phase of the cell cycle after treatment with high (15 Gy) dose of γ-radiation .Cells expressing HeLa-RhoAN19, expected to be deficient in RhoA activity, display higher senescence levels at lower doses of 0.5 and 5 Gy of γ-radiation, which correlates with their preferential arrest at G1 and S phases.These cells also showed increased apoptosis 48 and 72 h after exposure to the highest (15 Gy) dose of γ-radiation." -- -- -- -- -- -- -- -- Human L cellular senescence 26649141 Sen_G_0256 BRG1 834543 protein coding IMR-90 -- Aging Accelerate Knockdown//SA-β-gal activity assay "We ectopically expressed wild type BRG1 in IMR90 normal human fibroblasts. Indeed, there was a significant increase in SAHF formation in cells ectopically expressing BRG1 compared with controls. Consistently,other markers of SAHF such as formation of H3K9Me2 and mH2A1.2 foci were also induced by the ectopically expressed wild type BRG1.Expression of SA-β-gal activity, a hallmark of cellular senescence, was also induced by wild type BRG1 expression." BRCA1//p16//p21 --//Upregulation//Upregulation Knockdown//SA-β-gal activity assay//Western blot "Compared with shBRCA1 alone cells, expression of wild type BRG1 but not mutant BRG1 induced a more pronounced senescence phenotype as indicated by a significant increase in SAHF formation and expression of SA-β-gal activity.This correlated with an increased induction of p16INK4a and P21cip1." -- -- -- -- Human L cellular senescence 23438604 Sen_G_0257 ING1 3621 protein coding Fibroblast -- Aging Accelerate SA-β-gal activity assay ING1a induced the expression of both p16 and Rb when ectopically expressed in young fibroblasts. ING1a also induced senescence-associated β-galactosidase staining and cell cycle arrest at the G0/G1phase of the cell cycle after about 48 h of ectopic expression. ITSN2 Upregulation RT-PCR//BrdU assay//Knockdown//SA-β-gal activity assay "We measured the expression of ITSN2 in senescent cells after knocking down ING1a using siRNA. We found that knockdown of ING1a in senescent cells led to significant down-regulation of ITSN2 mRNA levels, further suggesting a role for ING1a in regulating ITSN2 expression .When ITSN2 was knocked down in ING1a-expressing cells, we found that EGFR internalization kinetics were similar to control GFP-expressing cells and were significantly restored when compared to the ING1a- expressing A431 cells. We next checked if loss of ITSN2 in ING1a-expressing cells affected the senescence phenotype. We found that senescence-associated β-gal staining was reduced significantly in these cells, and they showed increased proliferation when assayed using the BrdU incorporation assay. High levels of p16 were no longer induced by ING1a , suggesting that ITSN2 was indeed a direct mediator of ING1a-induced senescence signal. Since we previously noted higher levels of both ING1a and ITSN2 in senescent versus low passage fibroblasts." Rb-E2F -- Western blot "Since hypophosphorylated Rb is the Activate form that binds and inhibits E2F, we next asked whether the Rb in ING1a-expressing cells physically associated with E2F. Western blot analysis of immunoprecipitated E2F1 in these samples confirmed that E2F bound Rb, avidly in the presence of ING1a compared to the GFP-expressing cells." Human HL cellular senescence 23472054 Sen_G_0258 SIRT1 23411 protein coding HepG2 -- Hepatocellular carcinoma Prevent Knockdown//SA-β-gal activity assay//Colony formation assay//FACS analysis "The morphology of cells infected with shSIRT1 viruses was transformed into a larger, flattened shape compared to shScr and non-infected controls. Morphological changes in HepG2 cells were associated with a decrease in proliferation, measured by colony formation and alamarBlue? assays,more cells arrested in G1 and less in S phase of the cell cycle ，Their complete lack of growth was accompanied with an induction of cellular senescence as shown by enlarged cell size and expression of pH-dependent beta-galactosidase activity." -- -- -- -- -- -- -- -- Human L cellular senescence 23339189 Sen_G_0259 XRCC4 7518 protein coding Fibroblast -- Aging Prevent SA-β-gal activity assay//EdU assay Overexpression of both XRCC4 and Lig4 suppresses the onset of SIPS. -- -- -- -- -- -- -- -- Human L cellular senescence 27391797 Sen_G_0260 LIG4 3981 protein coding Fibroblast -- Aging Prevent SA-β-gal activity assay//EdU assay Overexpression of both XRCC4 and Lig4 suppresses the onset of SIPS. -- -- -- -- -- -- -- -- Human L cellular senescence 27391797 Sen_G_0261 TACC3 10460 protein coding MCF-7 -- Aging Prevent SA-β-gal activity assay//Knockdown "shRNA-mediated depletion of TACC3 resulted in a strong inhibition of MCF-7 cell proliferation, as evidenced by the progressive accumulation of cells arrested in G1and a decline in bromodeoxyuridine incorporation as a marker for DNA synthesis，Consistently, these cells developed several signs of a senescent phenotype, including a flattened and enlarged morphology and increased expression of senescence-associated βgal activity." -- -- -- -- -- -- -- -- Human L cellular senescence 20729911 Sen_G_0262 PTEN 5728 protein coding -- Prostate Prostate cancer Prevent SA-β-gal activity assay Ptenpc?/? mutants showed a strong senescence response as determined by SA-β-gal activity. -- -- -- -- -- -- -- -- Human HL cellular senescence 19690330 Sen_G_0263 AKT1 207 protein coding Human endothelial cell -- Aging Accelerate SA-β-gal activity assay "Long-term culture studies showed that constitutive activation of Akt significantly shortened the lifespan of the endothelial cells, whereas inhibition of Akt activity delayed senescence compared with mock-infected cells ,AktCA-transduced endothelial cells were flattened and enlarged, while mock- or AktDN-infected endothelial cells exhibited normal morphology and growth.Senescence-associated b-galactosidase activity was also increased in AktCA-transduced cells." -- -- -- -- p53-p21 Upregulation Western blot//Luciferase reporter assay "Expression of p53 and p21Waf1/Cip1, but not p16Ink4a, was elevated, while the level of phosphorylated Rb was decreased in AktCA-infected cells compared with mock infected cells, suggesting that Akt may induce growth arrest by upregulating p53 and p21，Aktinduced cell growth arrest was restored in p21-deficient MEF, suggesting that p21 is essential for Aktinduced growth arrest of these cells.Introduction of AktCA induced p53 promoter-driven luciferase activity compared with mock infection, but not luciferase activity driven by a promoter containing 15 copies of a similar sequence with mutation at critical positions (MG15),Ablation of p53 also lessened the decrease in the lifespan of AktCA-infected cells." Human L cellular senescence 14713953 Sen_G_0264 TAZ 6901 protein coding LN-229 -- Glioblastoma Prevent SA-β-gal activity assay//Knockdown "TAZ knockdown reduces the expression of CTGF and Cyr61 in these cells. Under this circumstance, more LN229 cells undergo senescence after irradiation. Notably, reduction of TAZ expression per se is not sufficient to mimic radiation in inducing senescence, although a modestly increased senescence frequency was observed. Therefore, inhibition of TAZ could facilitate tumor cells to undergo senescence after irradiation." -- -- -- -- Wnt Downregulation Western blot "To test if radiation-induced TAZ degradation is through inhibiting the Wnt signaling, we first examined the expression of βcatenin. Similar to TAZ, the protein level of β-catenin was also reduced progressively in response to irradiation in LN229 cells." Human HL cellular senescence 30542117 Sen_G_0265 NNT 23530 protein coding MSC -- Aging Prevent SA-β-gal activity assay "Replicative senescence in MSCs was clearly reduced by overexpression of NNT or NMNAT3 as determined by SA-β-gal staining.The proportions of SA-β-gal1 cells markedly decreased by 52.0% and 75.8% following overexpression of NNT and NMNAT3, respectively, compared with old control cells." -- -- -- -- -- -- -- -- Human L cellular senescence 27428041 Sen_G_0266 NMNAT3 349565 protein coding MSC -- Aging Prevent SA-β-gal activity assay "Replicative senescence in MSCs was clearly reduced by overexpression of NNT or NMNAT3 as determined by SA-β-gal staining， The proportions of SA-β-gal1 cells markedly decreased by 52.0% and 75.8% following overexpression of NNT and NMNAT3, respectively, compared with old control cells." -- -- -- -- -- -- -- -- Human L cellular senescence 27428041 Sen_G_0267 MUS81 80198 protein coding "SKOV3,A2780" -- Ovarian cancer Prevent SA-β-gal activity assay//Knockdown//Flow cytometry "The stable transduced shMUS81 SKOV3 and A2780 cells were arrested in G0/G1 phase ,and the percentage of S phase cells decreased obviously.Knockdown of MUS81 in SKOV3 and A2780 cells increased the emergence of premature senescence cells." -- -- -- -- -- -- -- -- Human L cellular senescence 27255997 Sen_G_0268 ATRAID 51374 protein coding ARPE?19 -- Aging Accelerate SA-β-gal activity assay//LDH activity assay "SA β-gal staining demonstrated that ~62.7% of cells overexpressing Apr3 were positive for SA β?gal, compared with 17.3% of the parent cells.Cell viability was then determined using an LDH activity assay and it was demonstrated cells overexpressing Apr3 exhibited decreased cell viability compared with the parent cells .Apr3 overexpression moderately inhibited cell proliferation by 34.5% compared with the parent cells as determined by a 3 [H]-thymidine incorporation assay." -- -- -- -- -- -- -- -- Human L cellular senescence 26934949 Sen_G_0269 CDKN2A 1029 protein coding MSC -- Systemic Lupus Erythematosus Accelerate SA-β-gal activity assay//Knockdown//Flow cytometry//BrdU assay There were less SA-β-gal-positive cells when p16INK4A expression was knocked down in MSCs from SLE patients. The cell proliferation assay showed that the proliferation rate of p16INK4A knockdown MSCs from SLE patients was restored to that of the normal MSCs. Cell-cycle analysis revealed that G1 phase arrest was reversed in p16INK4A-knockdown MSCs from SLE patients . -- -- -- -- -- -- -- -- Human L cellular senescence 22820504 Sen_G_0270 CDKN2A 1029 protein coding Human WMM1175 melanoma cell -- Melanoma Accelerate SA-β-gal activity assay "At 3 days post-induction the cells induced for p16INK4a expression had adopted characteristics of senescent cells, appearing enlarged and flattened. These cells were negative for the proliferation marker Ki67 and acquired SA-β-gal activity." CDK4//CDK6 Downregulation//Downregulation SA-β-gal activity assay "In the presence of ectopic CDK4 or CDK6 expression, p16INK4a did not promote cell enlargement, heterochromatic foci or SA-β-gal activity .These data confirm that the inhibition of both CDK4 and CDK6 kinase activity is required for p16INK4a-mediated cell cycle arrest and senescence." -- -- -- -- Human L cellular senescence 18843795 Sen_G_0271 DNMT1 1786 protein coding HDF -- Aging Prevent SA-β-gal activity assay//Western blot "Upregulation of DNMT1 was found to partially reverse the observed UVA-induced increase in SAβ-gal positive cells.DNMT1 expression attenuated the observed increase in p53, p21, and p16 expression following UVA irradiation." ZEB1 Binding Luciferase reporter assay//SA-β-gal activity assay//Western blot "Compared with the control vector, ZEB1 over-expression caused a significant increase in luciferase activity in the cells that were cotransfected with the WT and Mut2-4 reporter vectors. Conversely, cotransfection of ZEB1-expressing vector with the Mut1 reporter vector gave a level of luciferase activity that was equivalent to the control.ZEB1 up-regulation partially reversed the UVA-induced increase in SA-β-gal positive cells and upregulation of p53, p21, and p16 expression, while ZEB1 down-regulation caused the opposite effects to be observed." -- -- -- -- Human HL cellular senescence 29466247 Sen_G_0272 FOXA1 3169 protein coding HEC-1B -- Endometrial cancer Accelerate SA-β-gal activity assay//MTT assay "Restoring FOXA1 expression induced morphological features of senescence, including accumulation of granular cytoplasmic inclusions and elevated β-galactosidase activity.MTT and plate colony formation assays revealed that HEC-1B cells transiently transfected with a FOXA1 expression plasmid showed significant growth inhibition and limited colony formation compared with the cells transfected with the negative control vector." p16 Activation qRT-PCR "Following treatment with LY 294002, expression of the senescence-associated genes p16INK4a and p27kip1 was markedly increased, while PTEN gene expression was decreased." Akt Downregulation Western blot "Western blot analysis of the protein levels of p(Ser473)-AKT and AKT revealed almost complete inhibition of p-AKT expression in cells treated with LY 294002 at 1, 10 and 20 μM, with optimal inhibition achieved at 10 μM." Human L cellular senescence 27349269 Sen_G_0273 RAC3 5881 protein coding HEK293 -- Aging Prevent SA-β-gal activity assay RAC3 overexpression significantly reduced the number of cells having large and heterochromatic nuclei and the positive SA β-Gal-stained cells induced by H2O2 or rapamycin. -- -- -- -- -- -- -- -- Human L cellular senescence 26469953 Sen_G_0274 TP53 7157 protein coding HepG2 -- Hepatocellular carcinoma Accelerate MTT assay//SA-β-gal activity assay Both colony formation and cell survival were significantly reduced in the TP53+/Δ40 clones compared to the RI clones.The percentage of senescence-associated βgalactosidase (SA-β-gal)-positive cells was significantly higher in the TP53+/Δ40 clones compared to the RI clones. -- -- -- -- -- -- -- -- Human L cellular senescence 27980070 Sen_G_0275 ARHGAP18 93663 protein coding HUVEC -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis "After 48 h, ARHGAP18 overexpression resulted in a significant increase in large cells, defined as senescent based on size and flattened appearance,expression of SA-β-galactosidase (SA-β-gal) and of p21 expression." AP1 -- Western blot "ARHGAP18-senescent cells also prevented the phosphorylation of c-Jun, an essential component of the transcription factor AP-1, which has also been shown to regulate components of the SASP and for adhesion molecule expression on EC." NF-κB Upregulation Western blot "In the absence of TNFa stimulation, both EV and ARHGAP18-senescent cells showed basal levels of NF-jB expression. Following TNFa stimulation, EV cells showed increased expression of the RelA/p65 subunit of the NF-kB complex , whereas the ARHGAP18-senescent cells did not show this same upregulation." Human L cellular senescence 25407919 Sen_G_0276 PPARD 5467 protein coding HCAEC -- Aging Prevent SA-β-gal activity assay "The increase in SA-β-gal activity was significantly reduced in the presence of GW501561, a PPARδspecific ligand, demonstrating the involvement of PPARδin the inhibition of Ang II-induced premature senescence." SIRT1 Upregulation RT-PCR//Knockdown//SA-β-gal activity assay "When cells were exposed to 100 nM GW501516, the increase in SIRT1 mRNA was significant at 1 h and reached a maximum at 3 h.Whereas knockdown of SIRT1 by siRNA significantly reversed the effect of GW501516 on both ROS generation and cellular senescence in HCAECs following exposure to Ang II." -- -- -- -- Human L cellular senescence 23000914 Sen_G_0277 SIRT1 23411 protein coding HDF -- Aging Prevent SA-β-gal activity assay//Flow cytometry "SIRT1 adenovirus resulted in the marked upregulation of SIRT1 protein. Furthermore, cells overexpressing SIRT1 showed attenuated UVB-induced cell senescence, In addition, SIRT1 overexpression significantly upregulated cell growth and rescued cells from G1 cell cycle arrest." FOXO3α//p53 --//-- Western blot Total acetylation of p53 and acetylation of p53 at Lys382 were increased.SIRT1 overexpression decreased the UVB-induced acetylation of p53. These results suggest that overexpressed SIRT1 deacetylates p53 and FOXO3α to elicit its protective action against the UVB-induced senescence. -- -- -- -- Human L cellular senescence 25165029 Sen_G_0278 CDKN2A 1029 protein coding CPC -- Aging Accelerate SA-β-gal activity assay//Knockdown "Interestingly, SAβG staining showed decreased numbers of senescent cells in the experimental hCPCs infected with lentivirus expressing shRNA against p16INK4A." -- -- -- -- NF-κB Upregulation Western blot//RT-PCR "RT-PCR and Western Blot with cytokines and NFκB related genes was performed and showed upregulation in NFKB1, TBK1, RELA, IKBKB, and TICAM2. NFKB2 and HMOX1 showed 3~4-fold increased mRNA expression, while TLR3 showed almost 5.5-fold increased mRNA expression in the sh-p16INK4A infected aging hCPCs compared to the control hCPCs." Human L cellular senescence 29675777 Sen_G_0279 STIM1 6786 protein coding "PC-3,DU 145" -- Prostate cancer Accelerate SA-β-gal activity assay//Growth curve assay//Flow cytometry "The percentages of β-Gal staining (+) cells (senescent cells) were significantly higher in DU145 and PC3 cells overexpressing STIM1-YFP, ORAI1 and ORAI1-STIM1-YFP than in the corresponding controls.We observed a low growth rate in DU145-ORAI1, DU145-STIM-YFP and DU145-Orai-STIM1-YFP cells, indicating that STIM1 and ORAI1 have inhibitory effects on cell proliferation.We found that DU145 and PC3 cells overexpressing STIM1-YFP, ORAI1 and ORAI1-STIM1-YFP were enriched in the G0/G1 phase and that the percentage of these cells in the G2/M phase was significantly reduced." -- -- -- -- -- -- -- -- Human HL cellular senescence 26257076 Sen_G_0280 RTN4 57142 protein coding DU 145 -- Prostate cancer Accelerate SA-β-gal activity assay//Flow cytometry "RTN4 overexpressed cells were in G2/M phase but only 22.4% of control cells were in this stage, suggesting that cell cycle was blocked in G2/M phase (p < 0.05).Blue perinuclear staining in a small subset of RTN4 overexpressed DU145 cells was observed while the control group cells showed no visible staining." -- -- -- -- -- -- -- -- Human L cellular senescence 30480803 Sen_G_0281 YY1 7528 protein coding H460 -- Aging Prevent SA-β-gal activity assay//Knockdown "Cells transfected with YY1i exhibited phenotypic changes that resembled those observed in the cells undergoing replicative senescence.These changes include increased SA-β-gal staining, flattened cell morphology, and enlarged cell size." p16 Downregulation Knockdown//Western blot//CHIP "We used a p16siRNA vector (p16i) to knockdown the endogenous p16 expression [30]. Apparently, compared with the cells transfected with YY1i alone, co-transfection of cells with YY1i and p16i vectors failed to induce cell senescence.Western blotting assays demonstrated that the p16 protein expression was decreased upon overexpression of YY1, while it was enhanced by knockdown of the endogenous YY1 in NCI-H460 cells.The ChIP data revealed that YY1 was enriched at the p16 promoter (P3) upon the overexpression of YY1." -- -- -- -- Human L cellular senescence 18558095 Sen_G_0282 PIM1 5292 protein coding 2BS -- Aging Accelerate SA-β-gal activity assay PIM1 induces cell senescence. SA-β-gal activity staining experiments showed strong staining compared to the empty vector control and mPIM1K67M groups. CBX8 Downregulation CHIP//CCK-8 assay//EdU assay//Colony formation assay//SA-β-gal activity assay "To further verify whether endogenous CBX8 and PIM1 interact with each other, co-IP experiments were performed in extracts of senescent cells induced by RasV12 as PIM1 expression was upregulated in RasV12-induced senescent cells [9,26]. CBX8 was observed to co-immunoprecipitate with PIM1. In reciprocal immunoprecipitations, PIM1 was observed presenting in CBX8 immunoprecipitates PIM1 overexpression inhibited the growth of 2BS cells, while ectopic expression of CBX8 could obviously mitigate such growth inhibition by PIM1 as indicated in CCK8, EdU incorporation and colony formation assays. Furthermore, SA β-gal activity staining experiment showed that overexpressing CBX8 could make the percentage of stained cells decreasing as compared with that of cells with PIM1 overexpressing alone, which suggests that overexpression of CBX8 may effectively counteract PIM1-induced senescence." -- -- -- -- Human L cellular senescence 29763603 Sen_G_0283 GLIS2 84662 protein coding -- Kidney Aging Prevent SA-β-gal activity assay "Concordant with H3K9me3 content, senescence-associated lysosomal β-galactosidase activity was virtually absent in Ksp-creKif3af/-;Glis2+/mut kidneys but was clearly detected in Ksp-creKif3af/-;Glis2mut/mut double mutants and more intensely in Glis2mut/mut kidneys." -- -- -- -- -- -- -- -- Human L cellular senescence 27181777 Sen_G_0284 HTRA1 5654 protein coding ARPE-19 -- Aging Accelerate SA-β-gal activity assay There were more SA-β-galactosidase-positive cells among HtrA1-expressing ARPE-19 cells (55%) than among control cells (44.3%) after H2O2 treatment. -- -- -- -- p38 MAPK Activation Western blot//SA-β-gal activity assay "p38 MAPK is activated simultaneously with the HtrA1-enhanced cell senescence in MEFs.To confirm that HtrA1 activates cell senescence through the p38 MAPK cascade, we examined the effects of a p38 MAPK inhibitor, SB203580, on the induction of cell senescence by recombinant HtrA1,SB203580 completely abolished the HtrA1- stimulated expression of p21CIP1/WAF1 and p16INK4a. Similarly, induction of SA-β-galactosidase activity by recombinant HtrA1 was inhibited by SB203580,showing that HtrA1 enhances cell senescence through the p38 MAKP cascade." Human L cellular senescence 23623979 Sen_G_0285 MAML1 9794 protein coding B16 -- Melanoma Prevent SA-β-gal activity assay "ShMaml1-B16 cells exhibited a large, flat morphology indicative of senescence, which was confirmed by enhanced SA-β-gal staining." STAT1 -- Western blot Western blot analysis showed an increase in both the total and phosphorylated levels of STAT1 in the shMaml1-B16 cells. -- -- -- -- Human L cellular senescence 22864395 Sen_G_0286 MCL1 4170 protein coding Human glioma cell -- Glioma Prevent SA-β-gal activity assay//Knockdown "The MCL1-siRNA group showed decreased cell density, enlarged body of some cells with flat triangle, and an intense sense of grain, and vacuolation, which were the characteristics of cell senescence, and the positive expression rate of β-galactosidase was (14.26 2.1)% ." -- -- -- -- PI3K-Akt Downregulation Western blot "Compared with the blank and NC groups, the protein levels of MCL1, Bcl-2, PI3K, Akt, Bmi-1 and extents of PI3K and Akt phosphorylation increased remarkably in the IGF-1 group, and the protein levels of Bax and PTEN decreased significantly ; while the protein levels of MCL1, PI3K, Akt, Bcl-2, Bmi-1 and extents of PI3K and Akt phosphorylation decreased and that of Bax and PTEN elevated significantly in the IGF-1 + MCL1-siRNA group." Human L cellular senescence 30296359 Sen_G_0287 PINK1 65018 protein coding NPC -- Aging Prevent SA-β-gal activity assay//Knockdown "These results indicated that PINK1/Parkin mediated mitophagy was inhibited in NPCs by down-regulation of PINK1. Furthermore, SA-β-Gal staining results showed that NPCs suffered PINK1-shRNA transfection accompanying with H2O2 treatment had the highest percentage of SA-β-Gal-positive cells (blue), and staining intensity.However, down-regulating PINK1 alone didn't increase the percentage of SA β-Gal-positive cells and staining intensity comparing with the control group." Parkin -- Western blot "The expression of LC-3 II/I decreased and Parkin, P62 increased markedly in NPCs with PINK1-shRNA transfection." -- -- -- -- Human L cellular senescence 29890141 Sen_G_0288 CABLES1 91768 protein coding HUVEC -- Aging Accelerate BrdU assay//Flow cytometry//SA-β-gal activity assay "The EdU results showed that the Cables1 transfection reduced cell proliferation. Similar results were also observed by flow cytometry. The results suggested that HUVECs had a lower rate of proliferation (S: 3.00 ± 0.20%) in the Cables1 group than in the control group (S: 10.78 ± 0.9%). Cellular senescence was evaluated after Ang II treatment, and SA-Gal activity was found to be significantly increased in Cables1-transfected HUVECs compared with the control cells. " -- -- -- -- p21 Upregulation Western blot p21 activation was analyzed by Western blotting. The results showed that Cables1 significantly increased p21 expression compared with the control group. Human L cellular senescence 28118639 Sen_G_0289 SIRT1 23411 protein coding CEP -- Disc degenerative disease Prevent SA-β-gal activity assay "The overexpression of SIRT1 also decreased the population of G1 phase cells (p< 0.05), the percentege of SA-β-Gal-positive cells (blue), and staining intensity of the CEP cells." -- -- -- -- p53-p21 Downregulation Western blot "The overexpression of SIRT1 and inhibition of endogenous SIRT1 activity significantly influenced the p53/p21 pathway and, thereby, CEP cell senescence." Human L cellular senescence 26940203 Sen_G_0290 COX5B 1329 protein coding "MDA-MB-231,MDA-MB-468,MCF-7" -- Breast cancer Prevent SA-β-gal activity assay//Knockdown//Western blot//qRT-PCR//Transwell assay "The efficiency of knockdown was confirmed with mRNA and protein level analyses. We then explored the effects of COX5B depletion on cell proliferation and migration. The result showed that loss of COX5B inhibited both cell proliferation and migration in the three cell lines. In the process, cells with sh-COX5B became flattened and elongated after three passages, which were morphological transformation of cell senescence. Thus, a β-galactosidase staining assay was used to determine whether the cells undergo cell senescence. We found that more COX5B-depleted cells (25~45%) stained positively than control cells (7~16%), suggesting that loss of COX5B induced senescence." -- -- -- -- -- -- -- -- Human L cellular senescence 26506233 Sen_G_0291 ARID1A 8289 protein coding HPNE -- Pancreatic cancer Accelerate SA-β-gal activity assay//Knockdown "ARID1A knockdown had no effect on cell apoptosis as demonstrated by Annexin V/Propidium Iodide apoptosis assay, but accelerated cell cycle progression in HPNE/KRASG12D cells. Second, ARID1A knockdown significantly reduced the percentage of senescence-associated bgal staining-positive cells. Third, accelerated cell cycle progression and inhibited senescence were also observed in the stable HPNE/KRASG12D/shARID1A cells compared with HPNE/KRASG12D/shNC control cells." p16 Upregulation Western blot "The results showed that restored ARID1A expression led to increased expression of p16, promotion of cell senescence." -- -- -- -- Human L cellular senescence 28942143 Sen_G_0292 EZH2 2146 protein coding MKN28 -- Gastric cancer Prevent SA-β-gal activity assay//Flow cytometry//Knockdown "To further elucidate reasons why inhibiting EZH2 promoted DOX-induced cell proliferation inhibition, wecounted cells positive for SA-β-gal expression, and observed SAHF formation again. In MKN28 cells carrying mutant p53, siRNA and EGCG caused marked increase in positive level of SA-β-gal staining and SAHF and there was increase in percentage of G0/G1 phase cells.These results suggested that EZH2 depletion promoted DOX-induced senescence in MKN28 cells. " p53 -- Western blot "EZH2 depletion increased accumulation of p53 protein and protein of its target gene p21, in AGS cells under co-treatment." -- -- -- -- Human L cellular senescence 24738879 Sen_G_0293 SREBF1 6720 protein coding HDF -- Aging Accelerate Knockdown//SA-β-gal activity assay//Flow cytometry "SREBP-1 knockdown effectively attenuated all senescence phenotypes induced by H2O2.Similar and more remarkable results by overexpressing SREBP-1 on gain of senescence phenotypes, including SA-β-galactosidase, granularity (SSC), and delayed cell growth in addition to induction of lipogenic enzymes were obtained in young HDFs." -- -- -- -- -- -- -- -- Human L cellular senescence 20615871 Sen_G_0294 GRPR 2925 protein coding A172 -- Glioblastoma Prevent Knockdown//SA-β-gal activity assay//Flow cytometry GRPR knockdown reduces GBM cell proliferation.Silencing of GRPR increases significantly the percentage of senescent cells based on cell count using ImageJ. EGFR//p38//p16//p21 --//--//--//-- Western blot "We also observed a reduction of p38 and an increase in activation of EGFR as assessed by the content of pEGFR in GRPRi cells.GRPRi cells showed increased expression levels of p53, as well as p21 and p16,suggesting that senescence was enhanced in these cells through the activation of the p53 pathway." p53 -- Western blot//Knockdown "GRPRi cells showed increased expression levels of p53, as well as p21 and p16,suggesting that senescence was enhanced in these cells through the activation of the p53 pathway ." Human L cellular senescence 26780458 Sen_G_0295 CPEB1 64506 protein coding "U87,U251" -- Glioma Prevent Knockdown//SA-β-gal activity assay U251 and U87 cell growth was inhibited by CPEB1 knockdown. Senescent cells can be detected with an assay that detects senescence-associated β-gal activity. miR-101//p53 --//-- Luciferase reporter assay//qPCR "When the cells were individually cotransfected with miR-101 and pMIR-CPEB1-30-UTR-WT, there was a significant reduction in luciferase activity compared with cells transfected with the negative control, thereby confirming that miR-101 directly interacted with the 30-UTR of CPEB1 in glioma cells. It was gratifying to observe that miR101 could inhibit the expression of CPEB1, and that the suppression of miR-101 could enhance the expression of CPEB1.After the knockdown of CPEB1, the level of expression of p53 was decreased in the U251 cells but not changed in the U87 cells. Furthermore, it was discovered that the knockdown of CPEB1 induced the overexpression of p21, which is downstream of p53." -- -- -- -- Human L cellular senescence 23788032 Sen_G_0296 NRSN2 80023 protein coding SMMC-7721 -- Hepatocellular carcinoma Accelerate Cell viability assay//SA-β-gal activity assay "We found that when NRSN2 was overexpressed, the cellular viability of SMMC-7721 decreased and the senescent cells increased after doxorubicin treatment for 48 h ." Bcl-2 -- Western blot//qRT-PCR "Considering the increased cell viabilities and the anti-senescence/apoptosis effect after NRSN2 silencing, we examined the change of PI3K/Akt pathway, which plays an important role in tumor growth [11] and Bcl-2 family proteins, which function as a critical modulator in cell apoptosis .Meanwhile, the cleaved PARP and pro-apoptotic protein Bax was decreased, while the anti-apoptotic protein Bcl-2 was significantly increased after NRSN2 silencing." PI3K-Akt-mTOR//p53-p21 --//-- Western blot "Knockdown of NRSN2 led to increased (473)p-Akt levels in MHCC-LM3 cells. After silencing NRSN2 in Hep3B, we treated these transfected cells with doxorubicin, a chemotherapeutic agent which is commonly used to induce cell senescence , for 2 days and we surprisingly found that compared to the control, the levels of p53 and p21 were decreased." Human L cellular senescence 26055238 Sen_G_0297 SIRT1 23411 protein coding iPSC-Ecs -- Aging Prevent SA-β-gal activity assay "Overexpression of SIRT1 led to a significant reduction of cells entering senescence when compared to control groups. Blocking SIRT1 with the inhibitor Ex-527 led to a higher percentage of senescent cells in all groups, suggesting a contribution of endogenous SIRT1." -- -- -- -- -- -- -- -- Human L cellular senescence 26413932 Sen_G_0298 HRH4 59340 protein coding "MDA-MB-231,MCF-7" -- Breast cancer Accelerate SA-β-gal activity assay "Present results indicate that the negative effect on proliferation exerted by histamine in MDA-MB-231 cells was elated to a 2-fold increase in cell senescence. In addition, this effect was reversed by JNJ7777120 treatment and H4R agonists mimicked histamine effect, suggesting the involvement of H4R in histamine-induced cell senescence .In the same way, histamine was able to augment the percentage of MCF-7 senescent cells (15.7±1.3 vs. 7.8±0.4 in untreated, P<0.001), which was partially blocked by the treatment with JNJ7777120. Moreover, H4R agonists elicited a similar response, increasing cell senescence." -- -- -- -- -- -- -- -- Human L cellular senescence 21622113 Sen_G_0299 CDKN1A 1026 protein coding HUVEC -- Aging Accelerate SA-β-gal activity assay "When compared with the results of empty vector control, HUVECs transfected with p16 exhibited a rapid increase in the proportion of senescent cells, as assessed by SA-β-Gal staining on days 3 and 5. Cells transfected by p21 also showed accelerated senescence, but this occurred only after 5 days of incubation and was milder than the effect of p16 transfection." -- -- -- -- -- -- -- -- Human L cellular senescence 16243918 Sen_G_0300 CDKN2A 1029 protein coding HUVEC -- Aging Accelerate SA-β-gal activity assay "When compared with the results of empty vector control, HUVECs transfected with p16 exhibited a rapid increase in the proportion of senescent cells, as assessed by SA-β-Gal staining on days 3 and 5. Cells transfected by p21 also showed accelerated senescence, but this occurred only after 5 days of incubation and was milder than the effect of p16 transfection." -- -- -- -- -- -- -- -- Human L cellular senescence 16243918 Sen_G_0301 HIF1A 3091 protein coding "SH-SY5Y,SKNBE2c,SKNAS" -- Neuroblastoma Prevent SA-β-gal activity assay//Knockdown "We observed that the vehicle-treated NBL shHIF1A and shEPAS1 cells showed greater senescence compared to the vehicle-treated NBL shCTR cells. This suggested that the decreased HIF1A and EPAS1 expression in these NBL cells might make them more prone to go into senescence, independent of the ATRA treatment." -- -- -- -- -- -- -- -- Human HL cellular senescence 26057707 Sen_G_0302 EPAS1 2034 protein coding "SH-SY5Y,SKNBE2c,SKNAS" -- Neuroblastoma Prevent SA-β-gal activity assay//Knockdown "We observed that the vehicle-treated NBL shHIF1A and shEPAS1 cells showed greater senescence compared to the vehicle-treated NBL shCTR cells. This suggested that the decreased HIF1A and EPAS1 expression in these NBL cells might make them more prone to go into senescence, independent of the ATRA treatment." -- -- -- -- -- -- -- -- Human HL cellular senescence 26057707 Sen_G_0303 NOTCH1 4851 protein coding HUVEC -- Aging Prevent SA-β-gal activity assay "We examined the replicative lifespan of infected cells and found that up-regulation of Notch1 prolonged the lifespan of endothelial cells along with a decrease of senescence-associated bgalactosidase (SA-β-gal) activity and decreased expression of senescence-associated molecules such as p53, p21, and p16." -- -- -- -- Id-1-p16 -- Knockdown//SA-β-gal activity assay//Proliferation assay "To investigate whether Notch1 deletion induced endothelial cell senescence via a p16-dependent pathway, we co-infected human endothelial cells with the p16 shRNA and Notch1 shRNA vectors. Notch1 deletion led to premature senescence of mock-infected cells but not p16 shRNA-infected cells, suggesting that disruption of Notch1 promotes endothelial cell senescence via an Id1/p16-dependent pathway." Human L cellular senescence 24950189 Sen_G_0304 ROC1 830029 protein coding "HuH-7,HepG2,LM 6" -- Liver cancer Prevent SA-β-gal activity assay//Flow cytometry "In contrast, senescence started to occur at 72 h with continuous increase and reaching the peak at 120 h .SiROC1 induced the G2–M arrest, which occurred at 48?h post ROC1 silencing, and reached a peak at 96?h in HepG2 and Huh7 as well as LM6 cells (data not shown). Morphologically, when compared with control cells, ROC1-silenced cells were much larger in size with flattened shape, a feature of cell senescence." -- -- -- -- -- -- -- -- Human L cellular senescence 22935614 Sen_G_0305 PRKD2 25865 protein coding "U87,GM133" -- Glioblastoma Prevent SA-β-gal activity assay//Knockdown "Silencing of PRKD2 induced acidic senescence-associated β-galactosidase activity (SA-β-gal) in U87MG (64+10.4% positive) and GM133 (41+ 5.4% positive) cells. SA-β-gal activity was also observed in both primary glioma cultures (GBM2, Gli25) in response to PRKD2 silencing." -- -- -- -- p53 -- qPCR//Immunoblotting "Moreover, PRKD2-deficient cells showed a distinct reduction in the expression of c-Myc on mRNA and protein level. None of these alterations were observed in p53mut GM133 cells . mRNA expression of transcription factor E2F1 was significantly attenuated in p53wt U87MG and p53mut GM133 cells." Human L cellular senescence 24463355 Sen_G_0306 XRCC3 7517 protein coding NIH-3T3 -- Hypertension-induced left ventricular hypertrophy Prevent SA-β-gal activity assay "Consistent with the result of spontaneous DSBs, cellular senescence was induced to a greater degree in 3T3-241Met than in 3T3-241Thr." -- -- -- -- -- -- -- -- Human L cellular senescence 29626209 Sen_G_0307 SYT7 9066 protein coding H23 -- Lung cancer Prevent SA-β-gal activity assay "Moreover, more beta-gal positively stained cells were found in control cells compared with SYT7 overexpressing cells, suggesting that SYT7 inhibited cellular senescence." p16//p21//p53 Downregulation//Downregulation//Downregulation Western blot//Knockdown//qPCR "Knocking down the expression of SYT7 increased the mRNA levels of P16, P21 and P53, and vice versa. Consistently, Overexpression of SYT7 inhibited the protein level of P16, P21 and P53, and knocking down SYT7 increased the protein level of P16, P21 and P53." -- -- -- -- Human L cellular senescence 30647108 Sen_G_0308 PAK4 10298 protein coding "U87 MG,LN-18" -- Glioblastoma Prevent SA-β-gal activity assay//Knockdown "β3 knockdown also led to enhanced methylation of histone H3 in senescence-associated heterochromatin foci,increased γ-H2AX positive nuclei indicative of DNA double strand breaks,and accumulation of senescence-associted acidic β-galactosidase activity." p21 Downregulation Western blot//Knockdown//SA-β-gal activity assay "Silencing of β3 induced a marked increased in p21 protein expression for multiple glioblastoma cells,whereas p21 levels were unchanged or even decreased following β3 knockdown in various epithelial cancer cells." -- -- -- -- Human L cellular senescence 26297735 Sen_G_0309 DPP4 1803 protein coding WI-38 -- Aging Accelerate SA-β-gal activity assay//Western blot "As shown, markers p16 and p21 were elevated while SIRT1 levels declined in cells overexpressing DPP4, supporting the notion that DPP4 promoted cell senescence. In addition, overexpression of DPP4 in WI-38 cells elevated SA-β-gal activity and decreased 3 H-thymidine incorporation." -- -- -- -- -- -- -- -- Human L cellular senescence 28877934 Sen_G_0310 LRP8 7804 protein coding Smooth muscle cell -- Cardiovascular disease Prevent SA-β-gal activity assay//Knockdown "In contrast, smooth muscle cells isolated from the aorta or the carotid arteries of Lrp8?/? mice displayed very slow growth rate under the same culturing conditions with significant reduction in population doubling after the first passage. The number of senescence-associated β-galactosidase positive Lrp8?/? cells after 8 days in culture with 10% FBS (≈25%–30%) was higher than the ≈5% to 8% of senescence observed in Lrp8+/+ cells." CDC20-PP2A -- CHIP//Western blot "We used coimmunoprecipitation to analyze the CDC20-PP2A complex formation in Lrp8+/+ and Lrp8?/? smooth muscle cells and found that binding of the regulatory PP2A-B56α subunit to CDC20 does not require apoER2, while binding of the PP2A-C (catalytic subunit of PP2A) to CDC20 requires apoER2 expression . More importantly, while no direct interaction of CDC20 with apoER2 was detected, we observed binding of apoER2 to PP2AC in coimmunoprecipitation experiments with apoER2 antibodies. Since CDC20 interaction with PP2A and aurora kinase dephosphorylation are necessary steps for cell cycle exit,35,36 these data, together with the observation of a trend toward increased CDC20 level in Lrp8?/? smooth muscle cells, is consistent with the interpretation that apoER2 deficiency suppresses PP2A-C interaction with CDC20, thereby preventing its activation to cause cytokinesis impairment and cell cycle arrest." -- -- -- -- Human L cellular senescence 31412739 Sen_G_0311 CCNB1 891 protein coding "Capan‐2 PC,PC primary cell" -- Pancreatic cancer Prevent SA-β-gal activity assay//Knockdown//MTT assay "The staining results showed that cells in the blank, NC, and PFT‐α + shCCNB1 groups grew at a high density and were in a diamond shape with observable nuclear division. Cells in the normal and shCCNB1 groups grew at a low density, and part of the cells was in a triangle and enlarged shape with observable vacuolar degeneration, indicating cell senescence. Cells in the PFT‐α group grew at a high density with observable nuclear division. The result of β‐galactosidase staining showed that compared to that in the normal group, SI was significantly lower in other groups.. In comparison with the blank group, the shCCNB1 group had significantly decreased cell proliferation, whereas the PFT‐α group had significantly increased cell proliferation (p < 0.05) and the NC and PFT‐α + shCCNB1 groups exhibited no significant difference in cell proliferation (p > 0.05)." Bax//caspase‐9//Caspase‐3//p21 Downregulation//Downregulation//Downregulation//Downregulation Western blot//qRT-PCR "The mRNA and protein expressions of Bax, caspase‐9, caspase‐3, p21, and p53 were significantly lower, and the protein expression of p‐p53 was significantly lower in other five groups (p < 0.05)." p53 Downregulation Western blot//qRT-PCR "Compared with those of the normal group, the mRNA and protein expressions of CCNB1 and MDM2 were significantly higher, the mRNA and protein expressions of Bax, caspase‐9, caspase‐3, p21, and p53 were significantly lower, and the protein expression of p‐p53 was significantly lower in other five groups (p < 0.05)." Human L cellular senescence 30069972 Sen_G_0312 DEK 7913 protein coding CaSki -- Cervical cancer Prevent SA-β-gal activity assay//Knockdown//Flow cytometry//MTT assay "Most of the CaSki–DEK and CaSki–IκB cells displayed an intense blue staining, indicative of senescence, whereas only a few positively stained cells were detected in untransfected cells. As measured by microscopic image analysis, the percentage of positive staining was 83.25 +? 9.46% in CaSki–DEK cells and 76.47 +? 6.31% in CaSki–IκB cells,whereas the positive staining in CaSki–neo and untransfected cells was 23.13 +? 7.28 and 18.92 +? 6.46% respectively. The cell growth curve showed that, compared with untransfected and CaSki–neo cells,the proliferation activity of CaSki–DEK cells was inhibited in a time-dependent manner.In CaSki–DEK and CaSki–IκB cells, more cells were blocked in the G0/G1-phase, with a corresponding decrease in the G2/M-phase. The percentage of cells in the G0/G1-phase was significantly increased in CaSki–DEK and CaSki–IκB cells compared with control cells (P < 0.05)." NF-κBp65 Upregulation ELISA//Immunocytochemistry "The NF-κB activity determined by ELISA supported the findings observed by immunoblotting. CaSki–DEK and CaSki–IκB cells showed a significant increase in the activity of the NF-κB p65 subunit compared with control cells (P < 0.05). In accordance with this, in CaSki–neo and untransfected cells, NF-κB p65 was retained uniformly in the cytoplasm and only a mild-to moderate expression was observed by immunocytochemical staining." -- -- -- -- Human L cellular senescence 22390170 Sen_G_0313 HES1 3280 protein coding "Caco-2,SW48" -- Colorectal cancer Prevent SA-β-gal activity assay//Knockdown "We knocked down Hes1 in SW48 and Caco2 cells and observed significantly more beta-galactosidase expression in Hes1 knocked down cells, suggesting more senescence in the Hes1depleted cells." -- -- -- -- STAT3-MMP14 -- Western blot//qRT-PCR//Knockdown We observed that only MMP14 expression level decreased upon Hes1 knockdown at the mRNA level in Caco2 cells. We then found knock-down Hes1 lead to MMP14 protein level decrease in SW48 cells.Hes1 depletion resulted in a decrease of STAT3 phosphorylation and no change of total STAT3 in the Caco2 and SW48 cells. Human L cellular senescence 26650241 Sen_G_0314 STAT5A 100135887 protein coding MEF -- Aging Accelerate SA-β-gal activity assay//Cell proliferation assay "We show here that primary MEFs also undergo cell cycle arrest, with markers of cellular senescence when forced to express a constitutively Activate allele of STAT5A (caS5A) or RasV12 by retroviral gene transfer." -- -- -- -- JAK-STAT5//p53 Activation//Activation SA-β-gal activity assay//PML immunofluorescence//Proliferation assay "To investigate whether we could also trigger senescence by activating JAK/STAT5 signaling upstream of STAT5, we used a Tel/Jak2 fusion protein.54 Similar to caS5A,Tel/Jak2 was also sufficient to trigger senescence cell cycle arrest in MEFs .The process required p53 or the p53 activator p19ARF, because it was totally prevented in MEFs from p53 or p19ARF null mice." Human HL cellular senescence 20536843 Sen_G_0315 HGF 3082 protein coding EPC -- Aging Prevent SA-β-gal activity assay//Western blot "HGF and SOD, but not VEGF, significantly attenuated Ang II–induced senescence of EPCs as determined by senescence associated -galactosidase staining, although HGF and VEGF alone promoted EPC senescence. Moreover, HGF and SOD significantly inhibited the induction of p53, p21Cip1, and p16INK4a by Ang II." Rac1//PIP3//Akt --//Downregulation//-- Western blot "Whereas pretreatment with HGF, but not VEGF, prevented Ang II–induced rac1 binding to GTP at 60 minutes. HGF also prevented Ang II–induced rac1 translocation from the cytosol to the plasma membrane.we examined the PIP3 production, and, finally, we found that pretreatment with HGF significantly inhibited Ang II–induced PIP3 production, whereas VEGF did not inhibit it.Consistently, pretreatment with HGF,but not VEGF, significantly inhibited Ang II–induced phosphorylation of Akt in EPCs." -- -- -- -- Human L cellular senescence 19047582 Sen_G_0316 CBS 875 protein coding "HUVEC,HAEC,HDF" -- Aging Prevent BrdU assay//Knockdown//SA-β-gal activity assay "CBS depletion led to decreased cell numbers in HUVEC but not HDF; it also significantly reduced the rate of cell proliferation, measured by BrdU incorporation studies, but had no effect on the rate of apoptotic cell death. CBS knockdown also reduced the proliferative capacity of human aortic endothelial cells (HAEC), included as an additional control, included as an additional control.Both in HUVEC and HAEC, CBS knockdown led to a premature accumulation of cells staining positive for senescence associated ?-galactosidase (SA-?-gal)." -- -- -- -- -- -- -- -- Human L cellular senescence 23117410 Sen_G_0317 HMGA2 8091 protein coding ULM -- Uterine leiomyomas Prevent Knockdown//SA-β-gal activity assay "Cells treated with HMGA2 siRNA had significantly increased levels of β-Gal staining, indicative of cellular senescence than the cells treated with control siRNA." p16//p21 --//-- Western blot "In addition, we observed that silencing of HMGA2 in ULM led to upregulation of both p16 and p21." Akt -- Western blot//Knockdown "A dose-dependent upregulation of pAKT was observed by Western blot analysis along with an increase in HMGA2 levels. Next, using HMGA2 ULM, HMGA2 was silenced using siRNA. With decreased HMGA2 levels, decreased pAKT levels were observed ." Human HL cellular senescence 29790226 Sen_G_0318 GNAO1 2775 protein coding "GY-7703,SMMC-7721" -- Hepatocellular carcinoma Accelerate CCK-8 assay//Western blot//SA-β-gal activity assay//Knockdown "In the 96 h observation, we found that after 72 h of transfection, the cell proliferation was comparably faster in cells transfected with siGNAO1-1 and siGNAO1-2, in which the protein expression of GNAO1 was remarkably inhibited, than that in cells transfected with NC(P < 0.05).In β-gal staining assay, after 72 h of transfection, a lower proportion of senescent cells was observed in the group transfected with GNAO1 siRNA compared with that in the group of NC and NTC, indicating that down-regulation of GNAO1 inhibitedthe senescence of HCC cells." -- -- -- -- -- -- -- -- Human L cellular senescence 23984917 Sen_G_0319 BRG1 834543 protein coding SW 13 -- Retinoblastoma Accelerate SA-β-gal activity assay 16.8% of the cells transfected with BRG1 were β-galactosidase positive compared to only 1.4% with the control vector. p21/WAF1/SDI Upregulation Western blot "Western blotting showed that the expression of p21 was significantly down-regulated by inhibition of the BAF47 protein, while the expression level of p16 was not significantly changed in HeLa cells." -- -- -- -- Human L cellular senescence 14729964 Sen_G_0320 FOXO1 2308 protein coding "T98G,LN-18" -- Glioblastoma Accelerate Flow cytometry//MTT assay//Knockdown "The results revealed that the overexpression of FOXO1 arrested the cell cycle in the G0/G1 phase, suppressing cells from entering the S phase. The depletion of FOXO1 increased the colony forming ability in cell proliferation of the LN18 and T98G cells." SIRT1//p16 Downregulation//Upregulation CHIP//Western blot//qRT-PCR "A ChIP assay was performed with FOXO1 antibody, which revealed that the SIRT1 promoter region interacted with FOXO1.In LN18 cells overexpressing FOXO1, the expression of SIRT1 was suppressed.The opposite results were obtained in the FOXO1-depleted cells, which showed SIRT1 was increased and p16INK4a was decreased .The mRNA levels of SIRT1 and p16INK4a were also regulated by FOXO1, which suggested that FOXO1 regulated SIRT1 and p16INK4a at the transcriptional level." -- -- -- -- Human L cellular senescence 29207098 Sen_G_0321 FOXO4 4303 protein coding COLO 829 -- Melanoma Accelerate SA-β-gal activity assay Ectopic FOXO4 expression rendered Colo829 cells positive for SA-β-GAL activity. BRAFV600E//p21 -- Western blot//qRT-PCR//Luciferase reporter assay "We therefore wondered whether BRAFV600E could signal through JNK toward FOXO4 to promote senescence. To fully address this question, we first determined all possible JNK sites of in vitro phosphorylated FOXO4 by liquid chromatography–tandem mass spectrometry analysis.In vitro phosphorylation by JNK significantly increased detection of wild-type FOXO4 by these respective antisera, especially the newly discovered Thr223 and Ser226, whereas FOXO4-4A in which these residues are mutated to Ala was not detected. Together, these results indicate that BRAFV600E promotes JNK-mediated phosphorylation of FOXO4.BRAFV600Ecooperated with FOXO4 to induce p21cip1 expression, and in correlation with the induction of senescence, FOXO4 expression increased p21cip1 expression in Colo829 cells. Similar effects were observed on p21cip1mRNA expression determined by quantitative real-time PCR. Moreover, BRAFV600Eand FOXO4 expression resulted in a synergistic activation of a luciferase-reporter gene driven by the p21cip1promoter." -- -- -- -- Human L cellular senescence 20959475 Sen_G_0322 BMI1 648 protein coding GIST882 -- Gastrointestinal stromal tumor Prevent SA-β-gal activity assay//Knockdown//Colony formation assay//CCK-8 assay "The β-Gal activity assay was applied in order to detect cell senescence . In comparison with the control group, the percentage of senescent cells involved with the Bmi-1 shRNA group significantly increased (26.7% ± 3.9% vs. 17.2% ± 2.7%; P < 0.05).The results of the soft agar colony formation assay indicated that the number of colonies contained in the control group was closer to that in the NC group (9.5 ± 3.1 vs. 10.1 ± 2.2; P > 0.05). In comparison with the control group, the number of colonies in the Bmi-1 shRNA group were reduced significantly (9.5 ± 3.1 vs. 3.2 ± 1.5; P < 0.05)." p16//p14 Downregulation//Downregulation qRT-PCR//Western blot//Knockdown "The results of the RT-qPCR method for detection of the mRNA expressions of Bmi-1, p16Ink4A, p14ARF, cyclinD 1, and CDK4 in the GIST882 cells among the three groups are shown . These results showed no remarkable differences of Bmi-1 mRNA expression between the control and NC groups (P > 0.05). In comparison with the control group, the Bmi-1 mRNA expression in the Bmi-1 shRNA group was reduced significantly (P < 0.05). In comparison to the control group, both p16Ink4A and p14ARF mRNA expressions in the Bmi-1 shRNA group were markedly increased (both P < 0.05).. Bmi-1 protein expression in the Bmi-1 shRNA group was shown to be evidently lower than that found in the control group(P < 0.05). In comparison with the control group, both p16Ink4A and p14ARF protein expressions were markedly increased, while both cyclinD 1 and CDK4 protein expressions were significantly reduced in the Bmi-1 shRNA group (all P < 0.05)." -- -- -- -- Human L cellular senescence 29042141 Sen_G_0323 MEOX1 4222 protein coding HUVEC -- Aging Accelerate BrdU assay//SA-β-gal activity assay//Flow cytometry "Likewise, we observed a decrease in the proportion of S phase cells when MEOX1 or MEOX2 were expressed in HUVECs. MEOX1 had the greatest effect on cellular proliferation and MEOX2Q235E decreased the proportion of S phase cells to the same extent as wild-type MEOX2. This corresponds to an increase from 4.5% senescence to 9.8% and 10.9% senescence, when comparing untransduced cells to cells ectopically expressing MEOX1 and MEOX2, respectively." p21/WAF1//p16 Activation//Activation Luciferase reporter assay//Western blot "MEOX1 and MEOX2 induced greater than two fold activation of the luciferase reporter from the 2272 bp p21CIP1/WAF1 promoter, when compared to the empty vector control .Interestingly, the DNA binding deficient MEOX1Q220E and MEOX2Q235E proteins were able to activate this luciferase reporter to a level comparable to wild-type proteins .Compared to the EGFP control, we observed a three-fold increase in p21CIP1/WAF1 mRNA levels and more than a two-fold increase in p21CIP1/WAF1 protein levels 48 hours after adenoviral delivery of p53. Likewise, expression of MEOX1 or MEOX2 resulted in significantly increased p21CIP1/WAF1 mRNA expression.Comparable to MEOX2, MEOX1 activated the expression of a luciferase reporter from the 2272 bp p21CIP1/WAF1 promoter in HEK293 cells." -- -- -- -- Human L cellular senescence 22206000 Sen_G_0324 MEOX2 4223 protein coding HUVEC -- Aging Accelerate BrdU assay//SA-β-gal activity assay//Flow cytometry "Likewise, we observed a decrease in the proportion of S phase cells when MEOX1 or MEOX2 were expressed in HUVECs. MEOX1 had the greatest effect on cellular proliferation and MEOX2Q235E decreased the proportion of S phase cells to the same extent as wild-type MEOX2. This corresponds to an increase from 4.5% senescence to 9.8% and 10.9% senescence, when comparing untransduced cells to cells ectopically expressing MEOX1 and MEOX3, respectively." p21/WAF1//p16INK5a Activation//Activation Luciferase reporter assay//Western blot "MEOX1 and MEOX2 induced greater than two fold activation of the luciferase reporter from the 2272 bp p21CIP1/WAF1 promoter, when compared to the empty vector control .Interestingly, the DNA binding deficient MEOX1Q220E and MEOX2Q235E proteins were able to activate this luciferase reporter to a level comparable to wild-type proteins .Compared to the EGFP control, we observed a three-fold increase in p21CIP1/WAF1 mRNA levels and more than a two-fold increase in p21CIP1/WAF1 protein levels 48 hours after adenoviral delivery of p53. Likewise, expression of MEOX1 or MEOX2 resulted in significantly increased p21CIP1/WAF1 mRNA expression.Comparable to MEOX2, MEOX1 activated the expression of a luciferase reporter from the 2272 bp p21CIP1/WAF1 promoter in HEK293 cells." -- -- -- -- Human L cellular senescence 22206000 Sen_G_0325 ATM 472 protein coding HUVEC -- Aging Accelerate SA-β-gal activity assay//Knockdown "Furthermore, knockdown of ATM by siRNA suppressed increase in SA-β-gal-positive cells induced by H2O2." -- -- -- -- ATM-Akt-p53-p21 Activation Western blot//RT-PCR//SA-β-gal activity assay "To assess whether ATM phosphorylation was caused by H2O2-induced redox imbalance. Western blot analysis showed that pretreatment of endothelial cells with NAC blocked the stimulatory effects of H2O2 on the expression of ATM-S1981, Akt-S473, p53-S15 and p21 .Cells transfected with ATM siRNA showed reduced ATM expression with inhibition of Akt and p53 phosphorylation in addition to down-regulation of p21 expression .Moreover, down-regulation of Akt, p53, or p21 by siRNA also suppressed an increase in SA-β-gal-positive cells induced by H2O2." Human L cellular senescence 20639198 Sen_G_0326 ADIPOQ 9370 protein coding -- Liver Tumor Accelerate SA-β-gal activity assay//Knockdown "The number of SA-β-gal positive cells in liver tumor tissue from APN KO mice was markedly less compared with that from WT mice, indicating that adiponectin promotes cell senescence in tumor tissues." CXCL1 Upregulation qRT-PCR qPCR analysis further confirmed that CXCL1 was highly up-regulated in the adiponectin-treated SL4 cancer cells. p53//p16 Activation//Activation Immunohistochemistry//Immunofluorescence "p53- and p16-positive cells was detected in adiponectin treated co-cultured cells but not without adiponectin treatment, no expression was detected for p19 and p21 moleculars. Moreover, immunostaining of cocultured cells with PDGFRa (a marker of fibroblasts) confirmed the p53- and p16-positive cells are fibroblasts. Intervention experiments also showed p53 and p16 were involved in angiogenesis and tumor growth." Human HL cellular senescence 27092462 Sen_G_0327 ATF3 467 protein coding "HKC,SCC" -- Skin cancer Prevent SA-β-gal activity assay "In vivo, ectopic ATF3 promoted tumorigenicity of H-rasV12-expressing HKCs and SCC cells, producing aggressive tumours with reduced senescence as those caused by calcineurin/NFAT inhibitors. Conversely, Atf3 knockdown overcame the tumour-promoting effects of these compounds and restored senescence." p53 Downregulation Western blot//qRT-PCR "Functionally, ectopic ATF3 expression, at levels comparable to those occurring in SCCs from CsA-treated patients ,blocked expression of p53 and senescence-associated genes." -- -- -- -- Human HL cellular senescence 20485437 Sen_G_0328 RCC1 1104 protein coding hTERT-RPE1 -- Aging Prevent SA-β-gal activity assay//Immunoblotting "In contrast, SABG-negative and proliferating cells gradually prevailed in the hTERT-RPE1RCC1-V5 cultures, concomitant with increased interphase and mitotic markers and the decline in cyclin D1 expression." -- -- -- -- -- -- -- -- Human L cellular senescence 26864624 Sen_G_0329 PSMB5 5693 protein coding B-MSC -- Aging Prevent SA-β-gal activity assay//BrdU assay//Western blot "The number of BrdU+ cells was significantly increased by 14% following PSMB5 overexpression (p < 0.01 vs. GFP control),Meanwhile, overexpressing PSMB5 attenuated the reduction of Cyclin D1 and CDK4 proteins, raising their expressions by 56% and 23%, respectively.The SA-β-gal assay was applied to identify the aged hBMSCs by staining them blue. Enhanced proteasomal activity by PSMB5 overexpression significantly reduced the percentage of blue cells from 55 ± 3% to 40 ± 4%." -- -- -- -- -- -- -- -- Human L cellular senescence 24393841 Sen_G_0330 TBX2 6909 protein coding MCF-7 -- Breast cancer Prevent Clonogenic assay//Knockdown//SA-β-gal activity assay "We performed TBX2 siRNA knockdown in three breast cancer cell lines and after confirming knockdown we assessed effects on cell growth using clonogenic assays. We observed dramatic growth inhibition in all three cell lines.Finally, using senescence-associated β-galactosidase staining we could also show that knockdown of either TBX2 or EGR1 by transfection of NDRG1 into MCF7 cells that resulted in increased expression of Dec1, indicating that NDRG1 may contribute to the cell senescence observed after TBX2 inhibition." NDRG1 Downregulation Western blot//RT-PCR//Knockdown "The most consistently TBX2-regulated gene was NDRG1, which showed consistent upregulation after TBX2 siRNA in several breast cancer cell lines. We also observed NDRG1 upregulation both at the mRNA and protein level after induction of DN-TBX2 in MCF7 cells . Together, these data show that TBX2 represses multiple target genes with NDRG1 consistently upregulated after TBX2 inhibition." -- -- -- -- Human HL cellular senescence 20348948 Sen_G_0331 ZEB1 6935 protein coding SW480 -- Colorectal cancer Prevent SA-β-gal activity assay//Knockdown We found that ZEB1 knockdown increased the number of SA β-gal-positive cells and that Wnt3a inhibited basal SA β-gal activity in SW480-shCtl cells but not in SW480-shZEB1-A cells. DDK1//mutant p53//MDM2//CtBP//macroH2A1 Activation//Activation//Activation//Activation//Downregulation Knockdown//Western blot//CHIP//qRT-PCR "Accordingly, the transcriptional activity of serial deletions of the DKK1 promoter increased when the site at?490 bp was included.The ability of endogenous ZEB1 to directly bind to the DKK1 promoter was tested for the site at ?490 bp by ChIP assay.Furthermore, mutation of the CtBP binding sites in ZEB1 (ZEB1-CIDmut) hampered ZEB1’s repression of H2AFY,We found that blocking of CtBP activity in SW480 cells by MTOB induced senescence and upregulated H2AFY.We found that overexpression of DKK1 or addition of rhDKK1, but not rhDKK3, in mutant SW480 cells upregulated mutant TP53 and MDM2 expression in these cells . In turn, DKK1 knockdown downregulated both mutant TP53 and MDM2 and this downregulation was reverted by addition of rhDKK1, but not of rhDKK3. The downstream effect of DKK1 via mutant p53 was corroborated by the knockdown of the latter with a specific siRNA (siTP53). siTP53, but not siCtl, downregulated mRNA expression levels of MDM2 and CTBP while increasing those of H2AFY ." -- -- -- -- Human HL cellular senescence 27965283 Sen_G_0332 THRB 7068 protein coding "ARO,MCF-7,COS-7" -- Aging Accelerate SA-β-gal activity assay "We examined SA-β-gal activity and found that the number of positively stained cells was significantly higher in the irradiated cultures overexpressing wtTHRB than in those treated by any singular agent or by any other combination. Effect of wtTHRB-Ad on the accumulation of SA-β-gal-positive cells was more than additive in irradiated MCF-7 and COS7 cultures, and additive in ARO, Infection with mutTHRB-Ad significantly decreased the number of SA-β-gal-positive cells in irradiated ARO and COS7 cultures." p21//p16//Rb Upregulation//Upregulation//Downregulation Western blot "One hundred-twenty hours after irradiation, i.e. when growth inhibition and senescence-like changes have already taken place in the cultures, cultures infected with wtTHRBAd consistently displayed the decreased phosphorylation of Rb, suggestive of cell accumulation in the G1 phase. Concordantly, these cells also had elevated p21." -- -- -- -- Human L cellular senescence 17965546 Sen_G_0333 H1-3 3007 protein coding WI-38 -- Aging Accelerate BrdU assay//SA-β-gal activity assay//Cell morphological analysis "Concomitant with the decreases in histone H1 protein levels, the expression of N fusions caused severe growth defects in young WI-38 cells as revealed by the growth curves and the BrdU incorporation assay. N fusions also induced a large, flat morphology and increased the percentage of SA–β-gal–positive cells, suggesting that the ectopic expression of N fusions induced premature senescence in WI-38 cells." p53//p21//Rb --//Upregulation//-- Western blot//Cell transfection Cells expressing N fusions showed decreased levels of phosphorylated Rb and cyclin A and increased levels of p21 and phosphorylated p53. -- -- -- -- Human L cellular senescence 17158953 Sen_G_0334 TGFB1 7040 protein coding MEF -- Aging Accelerate Western blot//Immunofluorescence//SA-β-gal activity assay "Compared to physioxia, hyperoxia promoted senescence and triggered activation of TGF-β signaling.Activation of TGF-β signaling by oxidative stress was accompanied by increased miR-29 accumulation and accelerated reduction in the abundance of total H4K20me3.Inhibition of TGF-β signaling by inhibitor treatment and Smad4 depletion diminished miR-29 expression, attenuated the reduction in Suv4-20h1 and Suv4-20h2 expression and produced partial recovery of H4K20me3. Moreover, alterations in TGF-β signaling changed the progression of senescence and specifically altered H4K20me3 without affecting other histone modifications. Disruption of TGF-β signaling by E-616452 restored H4K20me3 levels,attenuated SA-β-gal staining and enhanced the Mki67 signals in Suv4-20h-depleted cells, suggesting that H4K20me3 is a downstream effector of TGF-β signaling. In addition, after miR-29 was destroyed by miRNA inhibitors, activation of TGF-β signaling did not down-regulate Suv4-20h and H4K20me3." miR-29//H4K20me3 Upregulation//Downregulation Western blot//qRT-PCR//SA-β-gal activity assay//Immunofluorescence "We performed histone modifications scanning in senescent cells. The results showed that H4K20me1, -me2, and -me3 exhibited prominent down-regulation in senescent mouse embryonic fibroblasts (MEFs). Accordingly, the expression levels of Suv4-20h1 and Suv4-20h2, the two major methyltransferases mediating H4K20me3, were also decreased during senescence. Furthermore, depletion of Suv4-20h1, Suv4-20h2 or both (designated as Suv4-20h) by shRNAs and treatment with selective Suv4-20h inhibitor A-19636 led to reduced H4K20me3 protein abundance and premature senescence.Meanwhile, disruption of the TGF-β pathway by E-616452 treatment mitigated the increase in miR-29 abundance and progression of senescence in cells with ectopic miR-29." -- -- -- -- Human L cellular senescence 29967491 Sen_G_0335 DGCR8 54487 protein coding IMR-90 -- Colorectal cancer Prevent SA-β-gal activity assay//Knockdown "shDGCR8 IMR90 cells also showed a significant increase in the number of cells with senescent‐associated beta‐galactosidase (SA‐Beta Gal) activity as well as nuclei with SAHFs (senescence‐associated heterochromatin foci), regions of facultative heterochromatin characteristic of senescent cells." p21 -- BrdU assay "Inactivation of DGCR8 in human and murine fibroblasts caused a modest, but reproducible increase in the protein levels of the cell‐cycle inhibitor p21CIP1. Also, a 3‐fold increase in p21CIP1 transcript and increased number of p21CIP1‐positive cells by immunofluorescence was observed in shDGCR8 human fibroblasts." -- -- -- -- Human HL cellular senescence 23773483 Sen_G_0336 HOPX 84525 protein coding "HBEC,Y-BE" -- Lung cancer Accelerate SA-β-gal activity assay//Cell morphological analysis "SA-β-Gal staining showed that HOPX transfectant cells displayed cellular enlargement and flattening as well as positivity for SA-β-Gal staining. Additionally, cells exhibited senescence-associated nuclear properties, including elevation of SAHF and accumulation of p-H2AX (gamma-H2AX, Ser139). Forced expression of HOPX in immortalized human bronchial epithelial cells Y-BE led to increased SA-β-Gal staining activity, while knockdown of HOPX in HBECs resulted in decreased SA-β-Gal positive staining, indicating that HOPX-mediated senescence is not restricted to malignant cells." -- -- -- -- Ras-MAPK//Akt Activation//Downregulation Western blot "We found that ectopic expression of HOPX resulted in activation of Ras in both HOPX-positive transfectants HOPX-3 and HOPX-V5-7, as revealed by Ras GTPase assay, which in turn led to activation/phosphorylation of ERK and p38, two major players of the MAPK pathway.Overexpression of HOPX led to inactivation of Akt and down-regulation of MDM2, a negative regulator of p53, as well as the p53 downstream target p21.Decreased expression of HOPX led to reversed effects on the Ras/MAPK pathway and the Akt pathway, with reduced phosphorylated levels of ERK and p38, as well as increased phosphorylated levels of Akt, MDM2, and down-regulation of p21." Human HL cellular senescence 25345926 Sen_G_0337 BHLHE40 8553 protein coding "HEK293,EC9706" -- Endometrial cancer Accelerate SA-β-gal activity assay//Colony formation assay//Western blot//Cell morphological analysis "We transfected DEC1 into EC9706 and confirmed that overexpression of DEC1 induces senescence by SA-β-Gal staining assay and the typically enlarged and flattened cell shape. We found DEC1 overexpression significantly suppressed the growth of EC9706 and HEK293, as measured by growth rate and colony formation assay." -- -- -- -- -- -- -- -- Human HL cellular senescence 22844531 Sen_G_0338 SIRT6 51548 protein coding HBEC -- Lung cancer Prevent Western blot//SA-β-gal activity assay//Immunofluorescence "Overexpression of SIRT6 significantly suppressed HBEC senescence after CSE exposure.Intriguingly, compared with control vector, a slight decrease in HBEC senescence was observed by SIRT6 overexpression even in the absence of CSE, especially when measured by means of SA-β-gal staining. Conversely, SIRT6 knockdown increased the percentage of senescent cells, indicating that intrinsic SIRT6 is not sufficient to completely inhibit senescence but has the ability to antagonize CSE-induced cellular senescence in HBECs." -- -- -- -- IGF-Akt-mTOR Downregulation Knockdown//Western blot "SIRT6 knockdown and mutant SIRT6 H133Y overexpression enhanced phosphorylation of IGF-1R accompanied by a modest increase in IGF-1R expression. Next, we examined phosphorylation of Akt and S6K. Consistent with IGF-1R phosphorylation, overexpression of SIRT6 clearly diminished Akt and S6K phosphorylation regardless of presence or absence of CSE. In contrast, SIRT6 knockdown and mutant SIRT6 H133Y overexpression induced Akt and S6K phosphorylation ." Human L cellular senescence 24367027 Sen_G_0339 GADD45G 10912 protein coding "SK-Hep-1,Hep3B,SMMC-7721" -- Hepatocellular carcinoma Accelerate SA-β-gal activity assay//RT-PCR "Approximately 50%‐70% of these cells ectopically expressing GADD45G were stained positively for senescence‐associated marker β‐galactosidase . Furthermore, mRNA levels of the senescence‐associated secretory cytokine, interleukin (IL)‐8, were also significantly elevated." -- -- -- -- JAK-STAT3 Downregulation Western blot//SA-β-gal activity assay "We found that the constitutive phosphorylation of Stat3 (Tyr705) was substantially inhibited by GADD45G expression in the human liver cancer cell lines, Sk‐Hep1, SMMC‐7721, and Hep3B.In Sk‐Hep1 cells, we found that levels of Tyr705‐phosphorylated Stat3 rapidly decreased, starting within 2 hours after GADD45G induction; coincidently, levels of phosphorylated Jak2 and tyrosine kinase 2 (Tyk2), the upstream activators of Stat3, were also severely down‐regulated.We extended our observation to two subclones (numbers 1 and 2) of mouse LPC‐H‐RasV12 cells, wherein GADD45G could significantly induce cell senescence.Western blot levels of phosphorylated Stat3, Jak2, and Tyk2 were also substantially decreased in these cells ." Human HL cellular senescence 23897841 Sen_G_0340 PRKN 5071 protein coding Airway epithelial cell Lung Chronic obstructive pulmonary disease Prevent Western blot//ELISA//SA-β-gal activity assay//Immunohistochemical staining "Senescence-associated-GLB1/β-galactosidase staining showed clear positive staining of airway epithelial cells in CS-exposed prkn KO mice but few positive staining cells were detected in CS-exposed wild-type mice.Immunohistochemical staining and western blotting of CDKN1A/p21 and CDKN2A/p16 (senescence-associated cyclin dependent kinase inhibitors) were performed. Both airway epithelial cells and alveolar epithelial cells in CS-exposed prkn KO mice demonstrated clear positive staining in both CDKN1A and CDKN2A . Consistent with the degree of cellular senescence, CXCL1 and IL1B protein levels were significantly increased in CS-exposed prkn KO mice compared to CS-exposed wild-type mice ." -- -- -- -- -- -- -- -- Human L cellular senescence 30290714 Sen_G_0341 CD40L 100286596 protein coding "A549,H460" -- Lung cancer Prevent Western blot//SA-β-gal activity assay//CCK-8 assay//Flow cytometry "We used pcDNA3.1+, pcDNA3.1+-CD40L-WT and pcDNA3.1+-CD40L-M to transfect CD40-positive NSCLC cell lines. CD40L expression was increased in the A549, A549/TR, A549/DDP and H460 cells 48 h after transfection.In addition, the culture cell size was enlarged in the A549/TR, A549/DDP and H460 cells, a trait associated with senescence. These data were confirmed with SA-β-gal staining, yet SA-β-gal staining was not observed in the A549 cells.CCK-8 assay data showed that CD40L-M expression significantly reduced cell proliferation.Flow cytometry data showed that the percentage of S phase cells was decreased and the percentage of G0/G1 phase cells was increased in the CD40L-M-expressed cell group compared with these percentages noted in the controls or negative cells,indicating that CD40L-M expression blocked cell cycle progression." -- -- -- -- ATM-Chk2 -- Western blot//SA-β-gal activity assay "We assessed p-Chk2 and p-Chk1 in CD40L-induced senescent A549/TR cells. Data showed that only p-Chk2 was significantly induced 48 h after transfection, indicating that the ATM/Chk2 pathway was activated in response to CD40L-M-induced senescence.We treated CD40L-M-transfected A549/TR cells with an inhibitor of ATM kinase, KU-55933. KU-55933 inhibited phosphorylation of the ATM target protein Chk2 and decreased SA-β-gal activity , suggesting that ATM/Chk2 mediated CD40L-M-induced senescence." Human L cellular senescence 29565449 Sen_G_0342 FOXO3 2309 protein coding MEF -- Aging Accelerate SA-β-gal activity assay//Knockdown We delivered the siRNA into MEFs and confirmed that transfection with this siRNA silenced circ-Foxo3 levels. We also delivered an siRNA targeting the linear mRNA of Foxo3 into the cells. The cells transfected with circ-Foxo3 siRNA showed less β-gal staining than the cells transfected with Foxo3 siRNA or the control oligo. Primary cardiomyocytes transfected with the siRNA also displayed lower levels of beta-Gal staining and circ-Foxo3 expression. -- -- -- -- -- -- -- -- Human L cellular senescence 26873092 Sen_G_0343 KNDC1 85442 protein coding HUVEC -- Aging Accelerate SA-β-gal activity assay//Flow cytometry "The levels of KNDC1 mRNA and protein in HUVECs at P4 were downregulated by gene silencing with KNDC1-siRNA1/2. Following repression of KNDC1 levels in HUVECs at P4, the size of the cells was similar to the size of P1 cells and the activity of SA-β-gal was decreased compared with that in NT-siRNA-transfected cells.The number of cells in G1 phase was decreased in cells transfected with KNDC1-siRNA." -- -- -- -- p53-p21-p16 -- Western blot The phosphorylation of p53 and the expression of p21 and p16 decreased significantly compared with NT-siRNA-transfected control cells. Human L cellular senescence 24788352 Sen_G_0344 HDAC2 3066 protein coding MCF-7 -- Aging Prevent SA-β-gal activity assay//Colony formation assay//Western blot//Knockdown "We found that knockdown of HDAC2, but not HDAC1, resulted in a G1 arrest.Consistent with the arrest in G1, cellular proliferation and colony formation were inhibited upon knockdown of HDAC2. We also found that HDAC2 affected colony formation in a dose-dependent manner because more effective knockdown of HDAC2 resulted in more marked growth inhibition .In contrast, knockdown of HDAC1 or tetracycline had no effect on cellular proliferation or colony formation. Furthermore, we found that knockdown of HDAC2 resulted in cellular senescence." -- -- -- -- -- -- -- -- Human HL cellular senescence 17409421 Sen_G_0345 DAO 1610 protein coding "U2OS,HepG2" -- Aging Accelerate SA-β-gal activity assay//Colony formation assay//Western blot//EdU assay "We observed that the percentage of SA-β-gal–positive cells was increased after the treatment with etoposide both in U2OS and HepG2 cells, knockdown of DAO partially restored etoposide-induced loss of proliferative capacity.we next tested the impact of inhibiting DAO activity on senescence using 6-chlorobenzo[d]isoxazol-3-ol (CBIO), previously characterized for the ability to inhibit enzymatic activity of DAO .CBIO suppressed the SA-β-gal activation and loss of proliferative capacity following etoposide treatment in both U2OS and HepG2 cells.CBIO was also effective in suppressing senescence of Hs68 cells, as judged by SA-β-gal, EdU incorporation proliferation assays, and immunoblot analysis, We observed that ectopic expression of DAO enhanced etoposide-induced senescence, although DAO overexpression in the absence of etoposide had no significant effect, indicating that DAO expression alone is not sufficient to induce senescence but accelerates senescence under stress conditions." -- -- -- -- -- -- -- -- Human L cellular senescence 30659069 Sen_G_0346 SIRT7 51547 protein coding WI-38 -- Aging Prevent SA-β-gal activity assay//Knockdown We found that shRNA depletion of SIRT7 from WI38 primary human fibroblasts led to substantial accumulation of senescent cells within days after SIRT7 depletion. Levels of the senescence marker p16 were also increased in the SIRT7-deficient cell cultures. -- -- -- -- -- -- -- -- Human L cellular senescence 29728458 Sen_G_0347 DPY30 84661 protein coding IMR-90 -- Aging Prevent Colony formation assay//SA-β-gal activity assay//SAHF//Cell morphological analysis//Knockdown "ShDPY30 IMR90 cells also contained DAPI-stained SAHFs, another characteristic for senescent human fibroblasts (Narita et al, 2003). Additionally, the overall morphology of the DPY30 knockdown IMR90 cells changed, towards a more rounded and flattened phenotype with enlarged nuclei. SA-β-galactosidase assays showed that >90% of the shDPY30 IMR90 cells expressed Activate SA-β-galactosidase. Further, in proliferation assays, growth curves, and colony formation assays, shDPY30 IMR90 cells arrested and stopped proliferating." ID -- Western blot//SA-β-gal activity assay "In ID1 and ID3 overexpressing cells, DPY30 knockdown reduced mRNA levels, but not proteins levels, of ID1 and ID3.the shDPY30 cells with ID1 or ID3 overexpression regained their fibroblast features and began to proliferate again, albeit not to the same degree as the IMR90 control cells, while the shDPY30 cells remained in a senescence-like state.?A reduced SA-β-galactosidase staining was observed in shDPY30 cells overexpressing either ID1 or ID3 (from 82 to 55 or 28%, respectively)." -- -- -- -- Human HL cellular senescence 23872946 Sen_G_0348 HRAS 3265 protein coding BJ -- Aging Accelerate SA-β-gal activity assay//Immunofluorescence "Doxycycline-induced H-RASV12?expression induces cellular senescence in normal BJ human fibroblasts.Few cells stained positive for (SA-β-GAL) activity during the proliferative phase at day 4 but SA-β-GAL positivity increased at day 12 in DOX-treated but not in untreated cultures, coinciding with the onset of growth arrest. By day 16 around 65 % of DOX-treated cells were SA-β-GAL positive compared to 15% of the control cells.?DOX stimulation increased the percentage of cells that showed intense H3K9me3 staining (90 % compared to 30 % in the control), appearing as SAHF or a more widespread nuclear staining ." p53 -- Western blot//SA-β-gal activity assay "RAS-induced SA-β-GAL activity was almost completely abolished in p53 knockdown cells, which is in good agreement with their continuous proliferation. Further, only a modest increase in the expression of p16 and H3K9me3 was observed in DOX-treated shp53 cells compared with shCtrl cells, suggesting that p53 depletion inhibited senescence induced by RAS." -- -- -- -- Human L cellular senescence 30526305 Sen_G_0349 Hsp27 39078 protein coding MRC-5 -- Aging Prevent SA-β-gal activity assay//qPCR//Knockdown "After 3 days of HSP27 knockdown, we observed significant increases in cells positive for senescence-associated β-gal (SA-β-gal) and significant up-regulation of pro-inflammatory cytokines IL-1α and IL-8." E2F4//p130 --//-- qPCR//Western blot//Immunofluorescence "By using qPCR, we found that the expression of E2F-4 was significantly increased upon HSP27 knockdown, HSP27 knockdown increased the expression of p130.We also confirmed strong increases of E2F-4 and p130 proteins by HSP27 knockdown using immunoblot analysis and immunocytochemistry." -- -- -- -- Human L cellular senescence 30166342 Sen_G_0350 XAF1 54739 protein coding HMVEC -- Aging Accelerate SA-β-gal activity assay//PI staining//Flow cytometry "Decreased cell proliferation in Doxo- or IR-treated cells was partially recovered by XAF1 downregulation. Repression of XAF1 levels in senescent cells caused morphological changes similar to young cells and a decrease in SA-β-gal activity. Additionally, XAF1 downregulation in premature senescent cells decreased the population of cells in the G1 phase and increased the population of cells in the S and G2/M phases. " -- -- -- -- p53 -- RT-PCR//Western blot//SA-β-gal activity assay "The Doxo- and IR-induced increase in p53 expression and activation was reduced in the XAF1 knockdown cells .By measuring the SA-β-gal activity, we demonstrated that p53 knockdown inhibited XAF1-induced cellular senescence but p16 knockdown did not." Human L cellular senescence 26802028 Sen_G_0351 IGFBP4 3487 protein coding MSC -- Aging Accelerate Cell apoptosis assay//Cell proliferation assay//Annexin V binding assay Treatment with anti-IGFBP4 and/or anti-IGFBP7 blocked the pro-senescence activity of CM-P10 even at the lowest concentration tested .It is to be noted that the simultaneous treatment with both anti-IGFBP4 and anti-IGFBP7 provided an enhanced reduction of senescence induced by CM-P10 on P1 MSC.treatment of CM-P10 with both anti-IGFBP4 and anti-IGFBP7 showed a significant reduction of the apoptosis level. CM-P10 treatment with anti-IGFBP4 or anti-IGFBP7 showed a significant increase of cell proliferation rates with respect to P1 MSC cultured with untreated CM-P10 at 72?h.the simultaneous addition of both rIGFBP4 and rIGFBP7 increased MSC senescence and induced apoptosis at the highest concentration assayed . -- -- -- -- -- -- -- -- Human L cellular senescence 24201810 Sen_G_0352 IGFBP7 3490 protein coding MSC -- Aging Accelerate Cell apoptosis assay//Cell proliferation assay//Annexin V binding assay Treatment with anti-IGFBP4 and/or anti-IGFBP7 blocked the pro-senescence activity of CM-P10 even at the lowest concentration tested.It is to be noted that the simultaneous treatment with both anti-IGFBP4 and anti-IGFBP7 provided an enhanced reduction of senescence induced by CM-P10 on P1 MSC.treatment of CM-P10 with both anti-IGFBP4 and anti-IGFBP7 showed a significant reduction of the apoptosis level.CM-P10 treatment with anti-IGFBP4 or anti-IGFBP7 showed a significant increase of cell proliferation rates with respect to P1 MSC cultured with untreated CM-P10 at 72?h.the simultaneous addition of both rIGFBP4 and rIGFBP7 increased MSC senescence and induced apoptosis at the highest concentration assayed . -- -- -- -- -- -- -- -- Human L cellular senescence 24201810 Sen_G_0353 ABI3BP 25890 protein coding ARO -- Aging Accelerate SA-β-gal activity assay//Flow cytometry "We first observed that SA-β-Gal positive cells,indicated by blue staining, were increased when ABI3BP is re-expressed.Secondly, immunofluorescence of HP1γ showed an expression pattern concentrated in DNA foci of cells expressing ABI3BP compared to controls which suggested that the re-expression of ABI3BP was associated with the nuclear changes that occur during senescence. Conversely in vector-transfected cells, the staining for HP1γ gave a weak signal and it was disperse through the nucleoplasm .Cell cycle distribution was analyzed by flow cytometry. G0/G1 arrest occurred in ARO cells expressing ABI3BP as indicated by an increase in the percentage number of cells at this phase." -- -- -- -- p21 Upregulation qPCR "P21 mRNA levels were 3-fold increased in ARO cells following ABI3BP re-expression . G0/G1 arrest occurred in ARO cells expressing ABI3BP as indicated by an increase in the percentage number of cells at this phase. Although cell cycle was not investigated at day 5, when senescence phenotype was marked observed, the cell proliferation marker Ki67 was down-regulated and the cell cycle inhibitor p21 was up-regulated at day 5." Human L cellular senescence 18559958 Sen_G_0354 PIM1 5292 protein coding 22Rv1 -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis "By the final passage (numbers 20–25), PIM1-expressing cells barely divided. The final-passage cells were found to have a flat and spread morphology and to be β-galactosidase (β-Gal) positive, consistent with the suggestion that they had undergone senescence." -- -- -- -- p53-p21 Activation SA-β-gal activity assay//qRT-PCR//MTT assay//Western blot "A marked change in the level of phosphorylation of these proteins occurs in late-passage PIM1-containing 22Rv1 cells and mirrors increases in the levels of the p21 protein . To examine the role of p21 in these cells, lentiviral vectors were used to transduce a Tet/ON-inducible shRNA directed at p21 (sh-p21) or a control sequence (sh-c) into 22Rv1 cells constitutively expressing PIM1 or a control vector. This shRNA was successful in decreasing p21 levels in control and PIM1-containing cells, but had no effect on the growth of 22Rv1 cells. In contrast, the growth of PIM1-containing 22Rv1 cells was markedly stimulated by decreasing the p21 levels. In these cells, decreased p21 levels correlated with a markedly lower level of γ-H2AX, staining of these late-passage PIM1–overexpressing cells with β-Gal shows that blocking p53 activity also prevents the induction of this marker of cell senescence by PIM1." Human L cellular senescence 20647331 Sen_G_0355 HEPACAM 220296 protein coding MCF-7 -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis "About 54.2% ( P ?10% of the PML signals colocalizing with telomeres whereas this was observed in only 3% of the PML/RARα cells.An increased expression of the p53, p16INK4a?and p21WAF1?proteins in PML-depleted cells with respect to control cells." -- -- -- -- Human L cellular senescence 26119943 Sen_G_0419 MET 4233 protein coding MDCK epithelial cell -- Tumor Accelerate Cell cycle analysis//BrdU assay//SA-β-gal activity assay "RON c5 and c8 were found to be largely arrested in the G0/G1?phase, in comparison to the broader cell-cycle distribution of MDCK and RON c1.BrdU staining indicated that RON c5 and c8 had significantly lower BrdU incorporation compared to that of MDCK and RONc1；RON c5 and c8 were undergoing senescence, in contrast to that of parental MDCK and RON c1 ." -- -- -- -- -- -- -- -- Human L cellular senescence 17588532 Sen_G_0420 PRKCA 5578 protein coding "HEK293,HCT116" -- Aging Prevent SA-β-gal activity assay Inhibition of cPKC or aPKC resulted in increased SA-β-gal staining as compared with control. -- -- -- -- Akt-FOXO3a-ROS-p53-p21 -- Immunostaining//Flow cytometry//Western blot "Treatment with triciribine markedly abolished PKC inhibition-mediated nuclear exportation of FoxO3a；We observed an increase in ROS accumulation, as shown by the rightward shift in DCF and ETH fluorescence in cells due to PKC downregulation；Accumulation of p53 and p21Cip1/WAF1 proteins was also observed in cells treated with G?o6983 or Ad-DN-PKCz, as compared with untreated control cells, suggesting that downregulation of cPKC and aPKC induces senescence through stabilization of p53 and transcriptional activation of p21Cip1/WAF1." Human L cellular senescence 28989024 Sen_G_0421 SIRT6 51548 protein coding WI-38 -- Aging Prevent SA-β-gal activity assay//Knockdown "SIRT6?knockdown (S6KD) cells have a strikingly shortened replicative lifespan, undergoing premature cellular senescence about ten population doublings before control cells, and show increased levels of senescence-associated β-galactosidase (SA-β-gal) staining." WRN -- Western blot//Flow cytometry "In both U2OS and IMR90 cells, SIRT6 knockdown significantly inhibited the association of WRN with telomeric chromatin." -- -- -- -- Human L cellular senescence 18337721 Sen_G_0422 PML 5371 protein coding IMR-90 -- Aging Accelerate SA-β-gal activity assay//BrdU assay "Cells arrested proliferation shortly after selection and remained arrested with features of cellular senescence, that is, a flat cell morphology and positive staining for the senescenceassociated β-galactosidase;PML induced a growth arrest with the characteristics of cellular senescence." -- -- -- -- Rb -- SA-β-gal activity assay//3H-thymidine incorporation assay "Cells expressing this mutant entered senescence in response to PML with higher efficiency than wild-type HDFs, according to a3 H-thymidine incorporation assay that measures cell cycle arrest, and according to the SA-β-gal assay that measures senescence,Therefore, the ability of E7 to by pass PML-induced senescence requires inactivation of the Rb tumor-suppressor pathway." Human L cellular senescence 14712214 Sen_G_0423 HMGB1 3146 protein coding "IMR-90,HCA2,HMEC,MEF" Embryo Aging Prevent Western blot//SA-β-gal activity assay//Knockdown "We depleted HMGB1 in human fibroblasts (IMR-90, HCA2) that reduced intracellular HMGB1 protein levels by >90%. HMGB1-depleted cells underwent senescence, as judged by arrested growth, senescence-associated heterochromatin foci (SAHF) formation (Narita et al., 2003), and SA-β-Gal activity.HMGB1 depletion also caused senescence in human mammary epithelial cells (HMECs) and MEFs, indicating the response was not restricted to human fibroblasts." p53//NF-κB --//-- SA-β-gal activity assay//Western blot//ELISA assay "Consistent with the IL-6 ELISA results, NF-κB activity was robust in HMGB1-overexpressing cells, but not HMGB1-depleted cells,senescence caused by HMGB1 overexpression or depletion was p53 dependent, as determined by SA-β-Gal activity, growth arrest (not depicted), and SAHF formation." -- -- -- -- Human L cellular senescence 23649808 Sen_G_0424 CCN1 3491 protein coding BJ -- Aging Accelerate BrdU assay//SA-β-gal activity assay//Immunostaining "Plication of CCN1 arrested cell proliferation within 3 days, as shown by stag-nant cell numbers and >50% decrease in 5-bromodeoxyuridine (BrdU)incorporation and Ki-67-positive cells when compared with bovine serum albumin (BSA)-treated controls ,CCN1-treated fibroblasts exhibited an enlarged and flattened cell morphology characteristic of senescent cells, and fourth, these cells expressed markers of senescence including SA-β-gal, p53 and p16INK4a." p53//NOX1//p38MAPK//ERK Activation//Upregulation//Activation//Activation Western blot//Knockdown//RT-PCR "Both p53 and p16INK4a proteins accumulated on CCN1 treatment, indicating their activation；CCN1 upregulated NOX1 expression in BJ cells,Knockdown of NOX1 by either of two shRNAs (#1 and #2) partially bypassed CCN1-induced growth arrest.Both kinases were activated by CCN1 in a biphasic manner (3 h and 24 h), and apocynin effectively abrogated ERK and p38 MAPK activation at both early and late phases" p16-pRb//Integrin --//-- Western blot//Knockdown//SA-β-gal activity assay "Both p53 and p16INK4a proteins accumulated on CCN1 treatment, indicating their activation；silencing of p16INK4a by either expression of shRNA (shp16INK4a) or its suppressor Bmi-1 partially restored CCN1-induced growth suppression .Pretreatment of cells with either soluble heparin or a function-blocking monoclonal antibody against α6 integrin inhibited CCN1-induced SA-β-gal expression, whereas an antibody against αvβ3 integrin had no effect, indicating the involvement of α6β1 integrin and HSPGs. Furthermore, the addition of a peptide that competitively binds α6β1 integrin specifically blocked CCN1-induced senescence, whereas a peptide with a two-residue substitution (T1-mut) that abrogated binding of α6β1 integrin was ineffective ." Human L cellular senescence 20526329 Sen_G_0425 TLR2 7097 protein coding IMR-90 -- Aging Accelerate Crystal violet assay//SA-β-gal activity assay Overexpression of TLR2 induced cell cycle arrest and an increase in the number of senescence-associated β-galactosidase (SA-β-Gal)–positive cells. p21//p16//p15INK4b//p53 --//--//--//-- Western blot "?Suppression of TLR2 and TLR10 resulted in a decrease in p21CIP1, p16INK4a, and p15INK4b?mRNA expression and a reduction of p53 protein levels." NF-κB//p38 MAPK//A-SAA --//--//-- Western blot//qRT-PCR "A significant enrichment in genes regulated by NF-κB in OIS (22) was observed in the transcriptome of control cells compared to TLR2-depleted cells ；We also observed a marked decrease in p38 MAPK phosphorylation in TLR2- and TLR10-depleted cells in OIS .Western blot of the conditioned medium from IMR90 ER:RAS cells showed an accumulation of A-SAAs, which was reduced following siRNA knockdown of SAA1 and SAA2, suggesting that A-SAAs are components of the SASP.Further mRNA analysis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) of AA1, SAA2, SERPINA3, PTGS2, STAT3, and IL-6R expression con-firmed these results, indicating that the expression of A-SAAs is highly induced during OIS and is dependent on TLR2 and TLR10." Human HL cellular senescence 31183403 Sen_G_0426 GOT2 2806 protein coding 8988T -- Pancreatic ductal adenocarcinoma Prevent SA-β-gal activity assay//Knockdown "GOT2 knockdown significantly elicited senescence in PDAC cells and also resulted in morphological changes, impaired PDAC growth and accumulation of cells in the G1 phase. Next, we found that overexpression of human GOT2 in PDAC cells can rescue the induction of senescence by GOT2 knockdown." p27 -- Knockdown//Western blot "When GOT2 was knocked down, p27 mRNA and protein levels were robustly induced in PDAC cells；to directly test whether p27 is required for the GOT2 knockdown-mediated senescence, we suppressed p27 expression using short interfering RNA (siRNA) in 8988T cells and assessed senescence upon GOT2 knockdown. Remarkably, silencing of p27 almost completely diminished senescence induction." -- -- -- -- Human L cellular senescence 29352139 Sen_G_0427 PDGFB 5155 protein coding HDF -- Aging Accelerate SA-β-gal activity assay//SAHF "Indeed they displayed a flattened morphology , an increase Senescence Associated β Galactosidase (SA-β-Gal) activity and an increase Senescence associated Heterochromatin Foci (SAHF)." p53 -- Knockdown//SA-β-gal activity assay//SAHF p53 knockdown strongly decreased the appearance of the senescence population in the PDGFB expressing cells as measured by loss of the SA-β-Gal positive cells and the decreased SAHF positive cells. -- -- -- -- Human L cellular senescence 23934686 Sen_G_0428 AR 367 protein coding PC-3 -- Prostate cancer Accelerate Cell cycle analysis//SA-β-gal activity assay "Longterm AR activation resulted in G1 growth arrest; the cells assumed flattened vacuolized morphology, suggestive of either autophagy or senescence. The levels of the principal autophagy mediator Beclin-1 [23,28] remained stable in the presence of DHT pointing to senescence. SA-β-Gal positivity increased from the background 4% to nearly 40% in the presence of DHT (P,0.0002), suggesting senescence." p53//p21//P63//Rb --//Upregulation//Downregulation//-- Knockdown//SA-β-gal activity assay//Western blot//RT-PCR "PC-3 cells are p53 and p16 null, RWPE-1 cells are p53 and Rb null. LNCaP cells express wild-type p53, but its levels were unchanged by DHT exposure;p21 was critical for AR-induced senescence since p21 silencing significantly reduced SA-βGal-positive population after DHT-treatment cells;p21 knock-down increased p63 protein and mRNA.AR activation also caused p63 exclusion from the nuclei,suggesting that nuclear DNp63 is important for blocking senescence. .P63 deficit likely contributed to the AR-driven senescence, since p63 knock-down elevated senescence in parental PC-3 cells.In PC3-AR and LNCaP treated with DHT, Rb phosphorylation visibly diminished." -- -- -- -- Human L cellular senescence 22403609 Sen_G_0429 PINK1 65018 protein coding HBEC Lung Aging Prevent SA-β-gal activity assay//Western blot PINK1 knockdown also enhanced CSE-induced mitochondrial ROS production and HBEC senescence. -- -- -- -- -- -- -- -- Human L cellular senescence 25714760 Sen_G_0430 PRKN 5071 protein coding HBEC Lung Aging Prevent SA-β-gal activity assay//Knockdown PARK2 knockdown enhanced HBEC senescence in response to CSE exposure as measured by SA-β-gal staining and expression of CDKN1A. -- -- -- -- -- -- -- -- Human L cellular senescence 25714760 Sen_G_0431 HAS2 3037 protein coding Fibroblast Lung Lung fibrosis Prevent Flow cytometry//BrdU assay//SA-β-gal activity assay//Knockdown "Deletion of HAS2 markedly suppressed the proliferation of fibrotic fibroblasts. Flow cytometry analysis revealed that HAS2 depletion significantly increased the proportion of cells in G1 phase.there was less BrdU incorporation in HAS2 siRNA transfected fibroblasts. Most interestingly, the proportion of cells with positive SA-β-gal staining was dramatically increased following HAS2 depletion." -- -- -- -- p27-CDK2-SKP2 -- Western blot//Knockdown "p27 protein levels were significantly increased in HAS2 deficient fibrotic fibroblasts at various time points after HAS2 siRNA transfection . Characterization of p27 in the presence of cycloheximide revealed that down-regulation of HAS2 increased p27 protein stability .CDK4 and cyclin B levels were also decreased in HAS2 deficient fibrotic fibroblasts.We observed a decrease (~ 30%) in SKP2 expression in HAS2 deficient fibrotic fibroblasts compared with control transfectants. We found that CDK2 protein levels were dramatically down-regulated upon HAS2 knock down in fibrotic fibroblasts ,which supported the hypothesis that HAS2 deletion-induced proliferative inhibition was through p27 accumulation and its effector pathway." Human L cellular senescence 26987798 Sen_G_0432 PLD2 5338 protein coding IMR-90 -- Aging Prevent SA-β-gal activity assay//Knockdown "We downregulated PLD2 using siRNA in proliferating IMR-90 cells (PDL 32) and found an increased rate of SA-β-gal staining in proliferating cells, compared with the control cells." CK2α -- SA-β-gal activity assay//Knockdown "Proliferating IMR-90 (PDL 32) and HCT116 cells were transfected with PLD2 siRNA, and a significant increase in the SA-β-gal activity was found in response to PLD2 knockdown, whereas co-transfection of cells with CK2α resulted in a significant decrease in the level of SA-β-gal staining." p53-p21 -- Knockdown//SA-β-gal activity assay//Western blot "In the wild-type HCT116 cells, significant increase in SA-β-gal activity in response to PLD2 knockdown was observed. However, p53- and p21Cip1/WAF1-negative cells did not show senescence；When the protein levels of p53 and p21Cip1/WAF1were determined by Western blot, the expression of p53 was found to be upregu-lated in both the wild type and p21Cip1/WAF1-/- cells treated with PLD2 siRNA, but not in p53-/-cells. The expression of p21Cip1/WAF1 was upregulated only in the wild-type HCT116 cells treated with PLD2 siRNA." Human L cellular senescence 25064843 Sen_G_0433 TP53 7157 protein coding IMR-90 -- Aging Accelerate SA-β-gal activity assay//Western blot "In TP53-deficient primary and tumour cell lines senescence decreased markedly and malic enzyme depletion lost its ability to induce this phenotype.By contrast, malic enzyme depletion did not cause cell death; it induced the expression of p53 target genes implicated in senescence but not apoptosis." ME1//ME2 --//-- Western blot "We knocked down TP53 in human osteosarcoma U2OS cells and normal diploid fibroblast IMR90 cells using short hairpin RNA (shRNA)，This led to a significant increase in messenger RNA levels of ME1 and ME2, accompanied by increased protein levels and total enzymatic activity of ME1 and ME2 ." -- -- -- -- Human L cellular senescence 23334421 Sen_G_0434 WT1 7490 protein coding "NCI-H522,NCI-H1437,NCI-H1568,NCI-H1650,NCI-H1975,NCI-H2126,NCI-H23,NCI-H358,NCI-H441,NCI-H460,NCI-H727,NCI-H1299,NCI-H2009,NCI-A549" -- Aging Prevent MTT assay//SA-β-gal activity assay "A significant decrease in the cell viability of cell lines harboring KRAS mutations was observed in response to WT1 loss.Mutant KRAS NSCLC cell lines adopted a senescence-like appearance in response to WT1 knockdown, and this was confirmed by staining with SA-βgal." -- -- -- -- Kras -- SA-β-gal activity assay//Tumor formation assay KrasG12D/+;Wt1Δ/Δ MEFs demonstrated a flattened morphology and stained positive for senescence-associated β-gal (SA-βgal). Wt1 suppression significantly decreased the size of tumors when compared with controls and to a level similar to that caused by suppression of Kras. Human HL cellular senescence 20972333 Sen_G_0435 WFDC1 58189 protein coding "HT-1080,WI-38,IMR-90" -- Aging Prevent Colony formation assay//qRT-PCR We found that overexpression of WFDC1 exerted an inhibitory effect on the colony formation efficiency of HT1080 cells；RNAwas produced from pre-senescent and senescent cells and the expression level of WFDC1 were measured by QRT-PCR. WFDC1 mRNA level was increased as well. -- -- -- -- -- -- -- -- Human HL cellular senescence 18842679 Sen_G_0436 ATF6 22926 protein coding HDF -- Aging Accelerate SA-β-gal activity assay//qRT-PCR//Knockdown "ATF6α silencing significantly reduced the number of SA-β-Gal positive-cells from about 60% in control cells to 25% in A TF6α silenced NHDFs. A small decrease of SA-β-Gal positive-cells was also observed upon IRE1α knock-down, but not upon PERK knockdown." -- -- -- -- -- -- -- -- Human HL cellular senescence 27563820 Sen_G_0437 PLK1 5347 protein coding MDA-MB-468 -- Breast cancer Prevent SA-β-gal activity assay//Western blot//EdU assay//DAPI staining PLK1 inhibition also resulted in senescence induction and p16 up-regulation in addition to p21 up-regulation in MDA-MB-468 cells. miR-200c//miR-141//BMI1//PRC1 Downregulation//Downregulation//Upregulation//-- qRT-PCR//Western blot//Knockdown "Our data indicated that indeed PLK1 knockdown results in up-regulation of miR-200c and miR-141, while PLK1 overexpression resulted in down-regulation of miR200c and miR-141 ；Our results indicated that indeed PLK1 overexpression leads to up-regulation of BMI1 and PRC1 activity." -- -- -- -- Human L cellular senescence 25505268 Sen_G_0438 NR2E1 7101 protein coding IMR-90 -- Aging Prevent Colony formation assay//BrdU assay//SA-β-gal activity assay//SAHF "NR2E1 expression resulted in an appreciable extension of replicative lifespan, as judged by cumulative population doublings and colony formation assays .A higher percentage of the NR2E1-expressing cells incorporated BrdU and a smaller percentage stained positively for SA-βGal activity, or showed evidence of senescence-associated heterochromatin foci；The expression of NR2E1 partially prevented the growth arrest and senescence observed upon RAS induction." CBX7 Upregulation qRT–PCR "We observed a direct correlation, suggesting that enhanced levels of NR2E1 associated with glioblastoma multiformeformation result in a concomitant increase in CBX7 expression ." -- -- -- -- Human HL cellular senescence 25328137 Sen_G_0439 SPRY1 10252 protein coding -- Thyroid gland Aging Accelerate SA-β-gal activity assay "Virtually all follicular cells from wild-type mice were strongly positive for SA-β-Gal activity, whereas only a minority of cells from knockout mice was faintly stained. together with increased Ki67 labeling of knockout follicular cells and increased proliferation in vitro,suggest that these cells bypass a program of cellular senescence engaged in the normal thyroid glands." -- -- -- -- NF-κB -- Western blot "Levels of IkBa, which is responsible for retention of NFkB dimers in the cytoplasm, were increased in the knockout thyroid.IkBa degradation was not impaired, IkBa levels after LPS challenging remained higher in thyroids from knockout mice when compared with wild-type mice." Human L cellular senescence 24270409 Sen_G_0440 HBP1 26959 protein coding 2BS -- Aging Accelerate BrdU assay//SA-β-gal activity assay//Growth curve assay "HBP1 played a role in growth suppression, as shown by BrdU incorporation assay and growth curve.HBP1 also induced premature senescence, as demonstrated by an increase of SA-β-gal staining." DNMT1 Downregulation Western blot//RT-PCR//Knockdown Exogenous HBP1 expression reduced DNMT1 protein (left) and mRNA (right) levels in both 2BS and WI-38 human fibroblasts.we used short hairpin RNA (shRNA) to knock down the HBP1 gene. An HBP1 knockdown increased DNMT1 protein and mRNA levels. -- -- -- -- Human L cellular senescence 23249948 Sen_G_0441 HRAS 3265 protein coding REF -- Aging Accelerate SA-β-gal activity assay We confirmed that oncogenic H-Ras induced senescence as scored by senescence-associated β-Gal activity. Myc -- SA-β-gal activity assay Ras-induced senescence was blocked efficiently by Myc. -- -- -- -- Human L cellular senescence 19966300 Sen_G_0442 WNT16B 51384 protein coding MRC-5 -- Aging Accelerate SA-β-gal activity assay//BrdU assay "When overexpressed in young MRC5 fibroblasts, WNT16B resulted in an increased percentage of cells positive for SA-β-Gal, a decrease in cell proliferation, and a decrease in bromodeoxyuridine incorporation." p21//p16 --//Upregulation Western blot Downregulation of WNT16B also prevented the accumulation of p21WAF1protein；WNT16B overexpression induced moderate p16INK4AmRNA and protein expression. PI3K-Akt Activation Western blot "AKT is also activated in WNT16Bexpressing MRC5 fibroblasts. Moreover, AKT activation was dependent on phosphoinositide 3-kinase (PI3K) activity and was abrogated through treatment with the specific PI3K inhibitor LY294002 ." Human HL cellular senescence 19951988 Sen_G_0443 MCM 4171 protein coding EPC2 -- Aging Accelerate Western blot "All of the examined MCM family members were found downregulated as EPC2 cells underwent senescence as documented by a reduced phosphorylation level of pRB protein；the MCM2-related fragment was expressed weakly in subconfluent EPC2 cells, where subpopulation cells undergoes spontaneous senescence in primary culture." -- -- -- -- -- -- -- -- Human L cellular senescence 19001876 Sen_G_0444 PFDN5 5204 protein coding "MCF 10A,H1299" -- Aging Accelerate SA-β-gal activity assay//Western blot "SA-β-Gal staining demonstrates that MM1 overexpression significantly increases the percentage of SA-β-Gal-positive cells, which is a hallmark of cell senescence, the protein level of cellular senescence marker PAI-1 is elevated." c-Myc//CD4//ΔNp63α//HERC3 Downregulation//Downregulation//--//-- Western blot//Knockdown//qRT-PCR The IB analysis demonstrates that c-Myc and its downstream target CDK4 are dramatically down-regulated by MM1.The results showed that up-regulation of MM1 induced by depletion of HERC3 also occurs in MCF-10A cells. Further study revealed that knockdown of HERC3 abrogates ΔNp63α-mediated down-regulation of MM1 protein levels. -- -- -- -- Human L cellular senescence 29880857 Sen_G_0445 TERF1 7013 protein coding Bone marrow cell "Bone marrow,Peripheral blood" Bone marrow failure Prevent SA-β-gal activity assay//Telomere length assay//Knockdown "To directly assess induction of senescence in vivo caused by TRF1 deletion, we used SA-β-gal actosidase staining directly on freshly isolated bone marrow cells.TRF1flox/floxMx1-Cre mice undergoing long-term Cre-induction/TRF1 deletion showed a dramatic telomere shortening of approximately 15 kb within 7-9 weeks of treatment in comparison with TRF1flox/floxMx1-wt mice after 13 weeks of treatment." p53//p21 --//-- Western blot//Immunostaining We found significantly greater p53 levels in pI-pC–treated TRF1flox/floxMx1-Cre mice compared with similarly treated TRF1flox/floxMx1-wt controls;a significant increase in p21 protein levels in the treated TRF1flox/floxMx1Cre group compared with the TRF1flox/floxMx1-wt controls. -- -- -- -- Human L cellular senescence 22932806 Sen_G_0446 CLCA2 9635 protein coding MCF-7 -- Aging Accelerate SA-β-gal activity assay We found an increase in positive staining for SA-β-gal in cells expressing CLCA2 compared with Mock-transfected cells. p53 -- qRT-PCR//Western blot//Northern blot "We selected CLCA2 for further analysis because CLCA2 was remarkably increased during the cellular senescence process as well as by the ectopic induction of wild-type p53 .We also confirmed the p53-mediated CLCA2 induction by quantita-tive RT-PCR, Northern, and Western blot analyses." -- -- -- -- Human HL cellular senescence 22431922 Sen_G_0447 DNMT3A 1788 protein coding HCT116 -- Aging Prevent SA-β-gal activity assay//Knockdown "We transfected HCT116 cells with DNMT3a siRNA plasmid, and we found that the number and intensity of SA-β-gal staining cells were increased at 1 lM Dox after DNMT3a was knocked down." p21 -- Western blot//qRT-PCR "We found that after transfection with DNMT3a siRNA, p21 protein level was upregulated.Transfection of DNMT3a expression plasmid downregulated p21 protein level." -- -- -- -- Human HL cellular senescence 20473858 Sen_G_0448 LMNA 4000 protein coding HCT116 -- Hutchinson-Gilford progeria syndrome Accelerate SA-β-gal activity assay "However, longerterm adenoviral overexpression of prelamin A for 7 days induced a significant increase in SAβGstaining." -- -- -- -- -- -- -- -- Human L cellular senescence 20458013 Sen_G_0449 ING1 3621 protein coding Hs68 -- Aging Accelerate SA-β-gal activity assay//BrdU assay "Cells treated with the agents noted above were tested periodically for SA-β-gal staining and BrdU incorporation,ING1a induced senescence by 36 hours and became maximal by 48 hours." -- -- -- -- p16-Rb Activation Western blot "Consistent with ING1a inducing changes similar to those seen in replicative senescence, Rb and p16INK4a levels increased to similar degrees in response to ING1a expression and replicative senescence." Human L cellular senescence 26439691 Sen_G_0450 ALOX15B 247 protein coding -- Prostate Prostate cancer Accelerate SA-β-gal activity assay "There was a noticeable increase in SA-βgal-positive glands in fl26 prostates compared with WT prostates, as previously observed.18 Important, there were also significantly more SA-βgal-positive glands in the prostates of Myc;LOX animals compared with Hi-Myc mouse prostates." p27//Rb//Rb1CC1 --// --// -- Western blot "Together, the results on p27 and Rb suggest that 15-LOX2-mediated tumor suppression in Hi-Myc model might involve cell senescence induction associated with molecular changes in these 2 molecules.The fact that increased Rb1cc1 expression was observed in 15-LOX2fl26 animals, which produce enzymatically Activate 15-LOX2 but not in WT or in 15-LOX2svb, which is enzymatically inActivate,18 suggests that 15-LOX2 metabolites may be mediating the Rb1cc1 induction." -- -- -- -- Human L cellular senescence 24732589 Sen_G_0451 PRKCI 5584 protein coding "MCF-7,T47D" -- Breast cancer Prevent SA-β-gal activity assay//Cell morphological analysis "PKCi depletion significantly increased the percentage of SA-β-gal-positive cells in both MCF7 and T47D cells. Cells that were SA-β-gal-positive showed an enlarged, flattened morphology." p21 -- Western blot//Immunostaining//SA-β-gal activity assay "As with MCF7 breast cancer cells, senescence occurred in the absence of a detectable DNA-damage response and was dependent on the presence of p21." -- -- -- -- Human HL cellular senescence 22120720 Sen_G_0452 SIRT1 23411 protein coding ADSC -- Aging Prevent SA-β-gal activity assay "Cells were treated with SRT1720, a specific SIRT1 activator. Consistent with expected result, SRT1720 decreased the number of SA-βgalimmunopositive cells." p53 Downregulation Western blot "p53 protein was decreased by SIRT1 overexpression (pb0.05), suggesting that SIRT1 negatively regulated P53 protein expression." -- -- -- -- Human L cellular senescence 29803744 Sen_G_0453 MAP2K3 5606 protein coding "184A1,MCF 10A" -- Breast cancer Accelerate SA-β-gal activity assay//MTT assay "Overexpression of MAP2K3 in 184A1 and MCF10A cells resulted in pronounced senescence of cells.PD rate of MAP2K3-expressing cells was decreased already after 3 days, and was halted by day 6, while the control cells continued to proliferate. When SA-β-galactosidase was measured, we observed that expression of MAP2K3 strongly increased its activity. Measurement of cell proliferation showed that expression of MAP2K3 had an inhibitory effect." p53//p38//pRb Upregulation//Upregulation//Upregulation Western blot "We measured the levels of p53, p38, pRB and Erk1/2 in cells transfected with MAP2K3 expression vector. We observed that enhanced expression of MAP2K3 led to increase of p53, pRB and p38 expression, but not Erk1/2." -- -- -- -- Human HL cellular senescence 21137025 Sen_G_0454 EGLN1 54583 protein coding HHUA -- Endometrial cancer Accelerate Cell morphological analysis//SA-β-gal activity assay "Seven clones out of HHUA transfectants, 8 of Ishikawa and 9 of HWCA showed flattened cytoplasm, frequently accompanied with multinuclei formation analogous to replicative cell senescence. A significantly increased fraction of acid-β-gal positive cells in the transected clones indicated the induction of cancer cell senescence by exogenous EGLN1 expression." p21//hTERT//HIF-1α Upregulation//Downregulation//Downregulation Western blot "In contrast, only the p21 CDK inhibitor was significantly upregulated in response to the EGLN1 transfection.Downregulation of hTERT was also observed in passage 5 of HHUA clone 3-2 and 122.As expected, both HHUA and Ishikawa cells silenced HIF-a expression in response to EGLN1 expression, followed by the suppressed expression of VEGF, which is a downstream transcriptional target of HIF-1a." -- -- -- -- Human L cellular senescence 16161047 Sen_G_0455 THBS1 7057 protein coding "HPAEC,HAEC" -- Aging Accelerate PI staining//Flow cytometry//MTT assay//BrdU assay//SA-β-gal activity assay//Cell morphological analysis "We found that TSP1 induced cell cycle arrest of HPAECs, characterized by increased numbers of cells in G1-G0 and decreased numbers of cells in S and G2-M; TSP1 significantly inhibited HPAEC proliferation, as detected by trypan blue exclusion,MTT assay , and BrdU incorporation; TSP1 exposure significantly increased the number of SA-β-Gal–positive HPAECs and human aortic endothe-lial cells, which showed concentration dependency,and elicited an enlarged and flattened cell shape characteristic of senescence ." NOX1//p53//p21 Upregulation//Upregulation//Upregulation Flow cytometry//qRT-PCR//Western blot//Immunostaining "FACS and mRNA analysis confirmed that TSP1 exposure significantly increased Nox1 abundance.We observed that Nox1 protein abundance was significantly lower in middle-aged TSP1?/?lungs compared to controls;We observed an increase in total p53 and p21cipin middle-aged mice.Moreover, we discovered that TSP1 exposure increased p53 abundance in HPAECs in a time-dependent fashion and increased p53 activation, as evidenced by its increased nuclear localization." -- -- -- -- Human L cellular senescence 29042481 Sen_G_0456 HIC1 3090 protein coding "PANC-1,BxPC-3,AsPC-1" -- Pancreatic cancer Accelerate MTT assay//SA-β-gal activity assay//Flow cytometry "Restoration of HIC1 function with 5-aza-dC treatment leaded to a reduction in cell proliferation, obvious cell senescence, cell cycle arrest and apoptosis, accompanied with acetylated p53 and p21WAF1 of Cip1 upregulation.Compared to untreated cells, cell proliferation of PANC-1, BXPC-3 and AsPC-1 treated with 5-aza-dC were significantly repressed . After treated with 5-aza-dC, the senescence positive rate of PANC-1, BXPC-3 and AsPC-1 cells were 43.6 ± 4.0, 69.9 ± 3.3 and 12.2 ± 2.2 %, all of which were significantly higher than in corresponding control group.Compared with control group, 5-aza-dC treated cells in G1/G0 phase were notably increased ." p53//p21/WAF1//SIRT1 Upregulation//Upregulation//Downregulation Western blot//MTT assay//SA-β-gal activity assay//Flow cytometry "After transfected with pCDNA3FlagHIC1, the acetylated p53 and p21WAF1/Cip1expression of PANC-1 cells significantly increased.After transfection with pCDNA3FlagHIC1, the cells demonstrated high-level expression of HIC1 protein and robustly downregulation of endogenous SIRT1 protein, which resulted in marked cell growth inhibition , cell senescence, cell cycle arrest and apoptosis." -- -- -- -- Human L cellular senescence 22552606 Sen_G_0457 IGFBP7 3490 protein coding "U87 MG,T98G" Glioma Aging Accelerate SA-β-gal activity assay "U87MG and T98G cultures treated with IGFBP7 (10 μg/ml) for 6 and 9 d, respectively, exhibited a larger number of senescence- associated β-galactosidase (SA-β-gal) positive cells compared with control cultures." -- -- -- -- -- -- -- -- Human L cellular senescence 21795858 Sen_G_0458 CD38 952 protein coding "NPC,5-8F" -- Nasopharyngeal carcinoma Prevent CCK-8 assay//Colony formation assay//SA-β-gal activity assay//Flow cytometry "The effect of CD38 on the proliferation of 5-8F cells was examined using a CCK-8 assay. The results demonstrated that CD38 promoted the proliferation of 5-8F cells .Acolony formation assay was performed. The results indicated that the high expression of CD38 clone number is increased, compared with the control group; The results demonstrated that the number of β-galactosidase-positive 5-8F/CD38 cells was reduced, compared with 5-8F/Vector cells. Additionally, it was demonstrated that CD38 overexpression reduced cellular senescence in NPC cells.This demonstrated that CD38 promoted S phase DNA replication and promoted the proliferation of NPC cells." CDK4//mitogen-activatedproteinkinase9//Cyclin D1 Upregulation//Upregulation//Upregulation Western blot "Upregulation of cell proliferation-associated genes CDK4, mitogen-activated protein kinase 9 and Cyclin D1 progressed the cell cycle from the G1 phase to the S phase resulting in increased cell proliferation." -- -- -- -- Human L cellular senescence 30535454 Sen_G_0459 NOTCH1 4851 protein coding "HMVEC,HAEC" -- Atherosclerosis Accelerate Cell morphological analysis//SA-β-gal activity assay "We tested the effect of enforced activation of Notch1 signaling on HAEC proliferation and observed that Notch activation resulted in significantly retarded cell growth and induced an enlarged, flattened and dendritic senescent morphology.Notch activation resulted in EC senescence since more senescent((β-gal﹢) cells were detectable from NICeGFP/HMVEC and NICeGFP/HAEC than GFP/HMVEC and GFP/HAEC." -- -- -- -- -- -- -- -- Human HL cellular senescence 23078884 Sen_G_0460 FGF21 26291 protein coding MSC Bone marrow Aging Prevent SA-β-gal activity assay//Immunostaining//Knockdown FGF21 siRNA treatment also markedly enhanced SA-β-gal-positivity in P4 BM-MSCs.Ki-67 staining revealed that the number of Ki-67-positive cells was significantly reduced in FGF21 siRNA-treated P4 BM-MSCs compared with control BMMSCs. p53//p21//Mfn2//p-Drp1 --//--//--//-- Western blot "We found that FGF21 siRNA treatment greatly reduced the protein level of FGF21 but upregulated the p53 and p21 protein level in P4 BM-MSCs,Silencing FGF21 with FGF21 siRNA elevated the level of Mfn2 and decreased the level of p-Drp1." AMPK -- Western blot//Knockdown Western blotting analysis showed that silencing FGF21 with FGF21 siRNA reduced p-AMPK and p-Drp1 and upregulated the Mfn2 level in P4 BM-MSCs. Human L cellular senescence 31178962 Sen_G_0461 TERT 7015 protein coding -- Lung Inflammation Prevent SA-β-gal activity assay//Immunostaining "β-Gal staining for cellular senescence was markedly increased in G3 TERT-null lung sections compared with control.We analyzed the levels of heterochromatin protein 1γ(HP1γ), a marker for senescence-associated heterochromatin foci, and found that immunoreActivate HP1γ was also markedly increased, with the level of HP1γin G3 TERT-/- being more than 2.5 times that in WT control animals." -- -- -- -- -- -- -- -- Human L cellular senescence 26518879 Sen_G_0462 GPX7 2882 protein coding "OE33,FLO-1" -- Oesophageal adenocarcinoma Accelerate Growth curve assay//SA-β-gal activity assay//Flow cytometry//Western blot//Colony formation assay "Using stable ( pcDNA-GPX7) and transient reconstitution models of GPX7 in OAC cell lines, we detected a significant reduction in growth rates in GPX7-expressing cells.Our analysis demonstrated that GPX7-expressing OAC cells (OE33 and FLO-1) had a significant increase in β-galactosidase staining (p≤0.01), as compared with control cells.About half of the control cells (Ad-CTRL) had proceeded tothe middle S phase after 4 h while only 32.4% ofGPX7-expressing cells (Ad-GPX7) proceeded to the early S phase." -- -- -- -- -- -- -- -- Human L cellular senescence 23580780 Sen_G_0463 HIC1 3090 protein coding TPC-1 -- Tumor Accelerate Flow cytometry//SA-β-gal activity assay//CCK-8 assay The number of cells in the G1 phase in the HIC1 transfected group (69.86 ± 1.22%) was slightly higher than in untransfected TPC-1 cells (62.36 ± 1.45%). The levels of β-galactosidase staining were also greater in the HIC1 overexpressing cells than the control TPC-1 cells. Cell proliferation in the HIC1 transfected group was significantly lower than in controls. SIRT1 Downregulation qRT-PCR//Western blot "The SIRT1 mRNA and protein expression levels were lowers in the HIC1 transfected group than in untransfected cells,showing that increased expression of HIC1 downregulated expression of the SIRT1 gene in TPC-1 cells." -- -- -- -- Human L cellular senescence 27793057 Sen_G_0464 HSF1 3297 protein coding MCF 10A -- Aging Prevent Growth curve assay//Cell morphological analysis "When we expressed HER2 in HSF1-depleted cells, a significant growth inhibition was observed, associated with a significant change in cells’ appearance, including enlarged, flattened morphology and extensive vacuolization reminiscent of senescence." -- -- -- -- -- -- -- -- Human L cellular senescence 20622894 Sen_G_0465 ERBB2 2064 protein coding MCF 10A -- Aging Accelerate Growth curve assay//Cell morphological analysis//SA-β-gal activity assay "When we expressed HER2 in HSF1-depleted cells, a significant growth inhibition was observed, associated with a significant change in cells’ appearance, including enlarged, flattened morphology and extensive vacuolization reminiscent of senescence.Expression of HER2 in shHSF1 MCF10A cells resulted in about 70% of β-gal-positive cells." p21 Upregulation Western blot There was a marked induction of p21 upon expression of HER2 in shHSF1 cells. -- -- -- -- Human L cellular senescence 20622894 Sen_G_0466 AKT1 207 protein coding BJ-T -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis//SAHF//Cell counting "Cells expressing activated AKT isoforms and H-RASV12 showed a significant increase in SA-β-GAL and cell size, indicating that AKT activation is sufficient to induce cellular senescence. The formation of senescence-associated heterochromatic foci (SAHF), a marker of RAS- and DNA damage-induced senescence;We found that AKT rapidly induced proliferative arrest." p53 Upregulation SA-β-gal activity assay//Immunostaining//Knockdown//DAPI staining A 45% increase in p53 synthesis was observed in the 30 min pulse period in myr-AKT-expressing cells compared with control cells.SA-β-GAL positivity was significantly reduced in BJ-T-myr-AKT/p53 stable knockdown cells as compared with control BJ-T-myr-AKT cells. -- -- -- -- Human L cellular senescence 21909130 Sen_G_0467 SUPT5H 6829 protein coding "RKO,HT29" -- Colorectal cancer Prevent SA-β-gal activity assay//Colony formation assay//Knockdown "A higher number of senescent cells was observed in SUPT5H-knockdown cells (shSUPT5H) compared to that observed in wild-type cells or negative control cells (shNSC);Colony formation assays showed that the inhibition of SUPT5H expression effectively suppressed the colony formation efficiency of transfected cells in both monolayer cultures and soft agar, compared to that observed in wild-type cells and negative control cells (p < 0.05)." hTERT -- Flow cytometry//Immunostaining "The results showed that inhibition of SPT5 expression by SUPT5H-specific shRNA attenuated hTERT promoter-driven GFP expression in RKO and HT29 cells transfected with hTERT promoter-driven GFP vector, whereas no obvious effect on CMV promoter-driven GFP expression was observed. Representative graphs of fluorescence-activated cell sorting (FACS) .These results indicated that SPT5 is essential for hTERT promoter activity and SUPT5H transcriptionally activated hTERT promoter-driven gene expression in human colon cancer cells." -- -- -- -- Human L cellular senescence 26418880 Sen_G_0468 CTNNB1 1499 protein coding "Ish-bcat#7,Ishikawa,Ish-tet#8" -- Endometrial cancer Accelerate Immunostaining//SA-β-gal activity assay "Some Ishbcat#7 cells with nuclear β-catenin expression demonstrated increase in size, and were more spread and flattened and multinucleated but not squamoid in appearance .After tetracycline treatment, there was an increase in numbers of senescence-like Ish-tet#8 cells similar to the case with Ish-bcat#7 cells. Four and five days after tetracycline treatment, SA-β-gal-positive cells were significantly increased with Ish-tet#8, but not the mock clones." Cyclin D1 Upregulation Western blot "In addition, increased expression of cyclin D but not PML was evident in Ish-bcat#7 cells." p53-p21 Upregulation Western blot "In addition, increased expression of p53, and p21WAF1 but not PML was evident in Ish-bcat#7 cells." Human L cellular senescence 15111320 Sen_G_0469 RAPGEF4 11069 protein coding EM1 -- Aging Prevent SA-β-gal activity assay//Western blot//Knockdown "The intensity of SA-β-Gal staining significantly increased in the EPAC2 or CALR siRNA-treated EM1 cells compared with the control cells. Furthermore, knockdown of either EPAC2 or CALR in EM1 cells suppressed the level of p53 but increased the level of p21." CALR -- Western blot//Knockdown "To determine whether knockdown of EPAC2 downregulated expression of CALR protein, the level of CALR in EPAC2-silenced EM1 cells was examined by immunoblot analysis. EPAC2 knockdown significantly suppressed CALR expression." -- -- -- -- Human L cellular senescence 25378661 Sen_G_0470 CALR 811 protein coding EM1 -- Aging Prevent SA-β-gal activity assay//Western blot//Knockdown "The intensity of SA-β-Gal staining significantly increased in the EPAC2 or CALR siRNA-treated EM1 cells compared with the control cells. Furthermore, knockdown of either EPAC2 or CALR in EM1 cells suppressed the level of p53 but increased the level of p21." CALR -- Western blot//Knockdown "To determine whether knockdown of EPAC2 downregulated expression of CALR protein, the level of CALR in EPAC2-silenced EM1 cells was examined by immunoblot analysis. EPAC2 knockdown significantly suppressed CALR expression." -- -- -- -- Human L cellular senescence 25378661 Sen_G_0471 MED12 9968 protein coding "PC-9,SPCA1" -- Lung cancer Prevent MTT assay//Colony formation assay//Histological staining//SA-β-gal activity assay "The MTT assay showed that MED12 KO clones proliferated significantly slower from day 2 compared with the MED12 WT or MED12 reconstitution single clones. This phenomenon was further confirmed by a colony formation assay.We found that MED12 knockout caused multinucleation in PC9 cells and restoring MED12 expression in MED12 KO cells was sufficient to avert multinucleation. A similar phenomenon was observed in SPC-A1 cells. Next, we investigated the cell fate of multinucleated cells and found that they exhibited senescence." -- -- -- -- -- -- -- -- Human HL cellular senescence 31072327 Sen_G_0472 GADD45A 1647 protein coding SCL-1 -- Skin cancer Accelerate SA-β-gal activity assay//Knockdown "Compared with the blank group,these nescence of tumor cells was postponed in the siGadd45a group (p< 0.05), while significantly improved in the Nutlin-3 group (p< 0.05). No significant difference showed in senescence among the blank, NC, and siGadd45a + Nutlin-3 groups (all p> 0.05). Cells transfected with siGadd45a had postponed senescence." p53 -- Immunostaining "The result showed that in the tumor tissues, the positive expression rate of mutant p53 was significantly higher than the normal group (all p< 0.05).Compared with the blank group, the positive expression rate of mutant p53 was significantly increased in the siGadd45a group (p< 0.05). The Nutlin-3 group presented decreased yellow granules, indicating that mutant p53 protein expression was decreased (p< 0.05)." -- -- -- -- Human L cellular senescence 29663367 Sen_G_0473 TERT 7015 protein coding hCMEC/D3 -- AIDS Prevent SA-β-gal activity assay "β-galactosidase was histochemically detectable in cells with silenced TERT (arrows), indicating that a partial loss of TERT expression is connected with accelerated hCMEC/D3 senescence." -- -- -- -- -- -- -- -- Human L cellular senescence 20139322 Sen_G_0474 SUV39H1 6839 protein coding "BEL-7402,SMMC-7721,MHCC97L,HuH-7" -- Hepatocellular carcinoma Prevent Colony formation assay//Cell proliferation assay//SA-β-gal activity assay//Knockdown "We showed that overexpression of SUV39H1 remarkably enhanced HCC cell clonogenicity, whereas SUV39H1 knockdown HCC cells reduced colony-forming ability;Knockdown of SUV39H1 significantly decreased cell proliferation and anchorage-independent growth of HCC cells;We observed an elevated senescence-associated lysosomal β-Gal activity in SUV39H1 knockdown cells." -- -- -- -- -- -- -- -- Human L cellular senescence 22991213 Sen_G_0475 BRCA1 672 protein coding PT-5006 -- Breast cancer Prevent SA-β-gal activity assay//Western blot//Knockdown "The beta-galactosidase assay demonstrated a higher degree of cell senescence in both BRCA1-knockdown adipose-derived stem cells and breast adipose-derived stem cells with the BRCA1 mutation；Western blotting also showed increased expression of p21, an established intermediate in the cell senescence pathway." -- -- -- -- -- -- -- -- Human L cellular senescence 30817646 Sen_G_0476 DUSP 17362377 protein coding RPMI8226 -- Myeloma Accelerate SA-β-gal activity assay//Western blot "The increase of DUSP evidently elevated the expression of P53, while reduced the level of TLR4. However, the block of DUSP reversed the effect on P53 and TLR4, indicating that DUSP participated in H2O2 induced myeloma cell line RPMI8226 aging and might regulate the level of TLR4." -- -- -- -- TLR4 Downregulation Western blot The increase of DUSP evidently reduced the level of TLR4. Human L cellular senescence 30280787 Sen_G_0477 SIRT6 51548 protein coding KM-HM_(31) -- Myeloma Accelerate SA-β-gal activity assay//Western blot "The increase of Sirtuin 6 apparently up-regulated the expression of P53,however, the block of Sirtuin 6 contributed to the opposite effect on P53 , indicating that Sirtuin 6 participated in CP-induced myeloma cell KM-HM_(31) aging." -- -- -- -- Hippo Downregulation Western blot "The increase of Sirtuin 6 apparently reduced the level of Hippo. However, the block of Sirtuin 6 contributed to the opposite effect on Hippo, indicating that Sirtuin 6 might regulate the level of Hippo." Human L cellular senescence 30402853 Sen_G_0478 LMNA 4000 protein coding MSC Umbilical cord blood Aging Accelerate Western blot//SA-β-gal activity assay//MTT assay "Immunoblots confirmed the over-expression of GFP-progerin and increased expression of p16INK4a, a marker of senescence in hMSCs;Induction of cellular senescence was also confirmed by the increase in SA-β-gal activity;MTT activity was decreased in response to progerin over-expression, indicating decreased proliferative activity." MCP-1//NF-?B Upregulation//Upregulation qRT-PCR//ELISA//Western blot//Immunostaining "Except for MCP-1, most cytokines that have been reported to increase in senescent fibroblasts were not increased in senescent hMSCs expressing progerin.The secretion of MCP-1 was also increased in the CM from progerin-expressing cells.The activation of NF-?B was also increased in response to progerin expression." -- -- -- -- Human L cellular senescence 29760459 Sen_G_0479 GATA4 2626 protein coding MSC Umbilical cord blood Aging Accelerate SA-β-gal activity assay//Knockdown The suppression of GATA4 in the progerin-expressing hMSCs significantly decreased SA-β-gal activity;The decrease in GATA4 also inhibited the induction of senescence mediated by CM from progerin expressing cells. MCP-1//NF-?B --//-- qRT-PCR//Western blot "GATA4 downregulation suppressed MCP-1 gene expression in progerin expressing hMSCs, suggesting that GATA4 regulates the SASP of hMSCs through MCP-1;The activation of NF-?B was also increased in response to progerin expression，however,the depletion of GATA4 decreased this activity." -- -- -- -- Human L cellular senescence 29760459 Sen_G_0480 DUSP22 56940 protein coding HS-1 -- Skin cancer Accelerate Western blot "Transfection of DUSP22 plasmid significantly elevated DUSP22 expression level in HS-1 skin cancer cells,inducing cell aging. Transfection of siRNA DUSP22 significantly depressed DUSP22 expression level in HS-1 skin cancer cells, decreasing expression levels of aging proteins P53 and P21." -- -- -- -- MAPK Activation Western blot "Transfection of DUSP22 plasmid significantly elevated DUSP22 expression level in HS-1 skin cancer cells, activating MAPK .Transfection of siRNA DUSP22 significantly depressed DUSP22 expression level in HS-1 skin cancer cells,inhibiting MAPK signal pathway." Human L cellular senescence 30536326 Sen_G_0481 HDAC6 10013 protein coding PDLSC Periodontal Ligament Aging Prevent MTT assay//Colony formation assay//SA-β-gal activity assay//Cell morphological analysis//BrdU assay "The results indicated that PDLSCs with HDAC6-inhibited by Rocilinostat or Tubastatin A displayed significantly lower proliferation rate compared with cells treated with DMSO as a control.Such inhibitory effect was further demonstrated using colony formation assay, as limited crystal violet staining of stable clones observed in HDAC6-inhibited cells versus control cells;Characteristic morphologies of senescence, including elevated SA-β-gal activity, enlarged and flattened cell size, and accumulation of granular cytoplasmic inclusions could be observed in Rocilinostat or Tubastatin A treated cells rather than control cells;Moreover, reduced BrdU incorporation further supported the pro-senescence action of Rocilinostat and Tubastatin A ." p27 Downregulation Western blot "We overexpressed HDAC6 and found significantly decreased p27Kip1 acetylation upon HDAC6 overexpression.Moreover, ectopic expression of HDAC6 also resulted in down-regulation of p27Kip1 proteins." -- -- -- -- Human L cellular senescence 27562218 Sen_G_0482 NAMPT 10135 protein coding -- Aortas Human Thoracic Aortic Aneurysm Disease Prevent SA-β-gal activity assay "However, in SMC-Nampt deficient mice there was SA-β-gal activity throughout the ascending aorta, arch,and proximal descending thoracic aorta after 7 days of Ang II infusion." p16 -- Western blot This revealed a 6.1-fold increase in the abundance of p16-expressing cells in the media of the ascending aorta of Ang II-infused SMC-Nampt KO mice relative to Ang II-infused control mice and a 4.7-fold increase in the thoracic aorta. -- -- -- -- Human L cellular senescence 28356339 Sen_G_0483 VDR 7421 protein coding MSC Femoral head Aging Prevent SA-β-gal activity assay//Cell proliferation assay "1,25D3 treatment of hMSC leads to a significant inhibition of cell proliferation after 48 h (17% reduction;p,0.01) and 72 h (27% reduction; p,0.001);In stimulated hMSC(gray bar) a significant reduction of β-galactosidase staining was observed compared to unstimulated cells (black bar) (p,0.001)." p16 Downregulation qPCR "The gene expression of the senescence-associated genes cyclin dependent kinase inhibitor 2B (P15), cyclin-dependent kinase inhibitor 2A (P16), cyclin-dependent kinase inhibitor 1A (P21) and proteasome 26S subunit, non-ATPase, 9 (P27) in hMSC cultured with or without 1,25D3 for four passages led to a significant 0.4-fold(p,0.05) downregulation of P16 gene expression." -- -- -- -- Human L cellular senescence 22242193 Sen_G_0484 LMNA 4000 protein coding Primary dermal Fibroblast Skin Aging Accelerate SA-β-gal activity assay "These analyses demonstrate that cells expressing progerin display a progressive, passage-dependent increase in the percentage of senescent cells over a time frame which spanned passages 5–10 compared to control cells ,which levels off after passage 10." -- -- -- -- -- -- -- -- Human L cellular senescence 18363904 Sen_G_0485 BMP4 652 protein coding A549 -- Lung cancer Accelerate Cell morphological analysis//SA-β-gal activity assay "A change in morphology was apparent after 2 wk of treatment. The slower-growing cells were larger, flattened, and more granular than untreated cell;A fourfold increase in S-A-β-gal activity was seen after 17 days of BMP4 treatment;Laddering was reduced in untreated A549 cells grown for 17 days in low-serum conditions making them essentially quiescent and dramatically reduced in cells treated with BMP4 for 17 days and expressing the senescence biomarker SA-β-gal." PERK//VEGF//Bcl2 Downregulation//Downregulation//Downregulation Western blot "A decrease in proliferative and angiogenic phenotypic markers compared with untreated cells cultured under parallel low serum conditions, as shown by decreased p-ERK and VEGF expression, respectively.The expression of survival factor Bcl2 is lower in BMP4-treated cells, suggesting an increased potential for apoptosis in the senescent population." -- -- -- -- Human L cellular senescence 12959928 Sen_G_0486 E6 1489078 protein coding SiHa -- Cervical cancer Prevent Cell morphological analysis//SA-β-gal activity assay//Knockdown "The depression of E6 caused the SiHa/16AS cells to cease proliferation and adopt a flattened and enlarged appearance, suggesting that the cells efficiently underwent senescence.The colonies arising from the mock-transfected showed faint background staining, whereas almost all of the cells transfected with 16AS displayed intense blue staining indicative of SA-β-gal activity and senescence." -- -- -- -- p53-Rb -- Western blot "Downregulation of HPV-16 E6 and E7 by 16AS transfection resulted in remarkably elevation of p53 expression in SiHa cells. Furthermore, the introduction of 16AS led to an obvious increase in the levels of hypophosphorylated p105Rb." Human L cellular senescence 17586029 Sen_G_0487 E7 1489079 protein coding SiHa -- Cervical cancer Prevent Cell morphological analysis//SA-β-gal activity assay//Knockdown "The depression of E6 caused the SiHa/16AS cells to cease proliferation and adopt a flattened and enlarged appearance, suggesting that the cells efficiently underwent senescence.The colonies arising from the mock-transfected showed faint background staining, whereas almost all of the cells transfected with 16AS displayed intense blue staining indicative of SA-β-gal activity and senescence." -- -- -- -- p53-Rb -- Western blot "Downregulation of HPV-16 E6 and E7 by 16AS transfection resulted in remarkably elevation of p53 expression in SiHa cells. Furthermore, the introduction of 16AS led to an obvious increase in the levels of hypophosphorylated p105Rb." Human L cellular senescence 17586029 Sen_G_0488 PEA15 8682 protein coding HDF Foreskin Aging Accelerate SA-β-gal activity assay//Western blot//Immunostaining//Knockdown//Cell proliferation assay "Nuclear translocation of pErk1/2 by siPEA-15 was accompanied with significant reductions of senescence markers in HDF old cells; over 10% decrease of SA-β-gal activity, and 50% decrease of p53 and p21WAF1expressions.Furthermore, treatment of old cells with siPEA-15 significantly reduced senescence markers such as PML body formation, and expressions of 53BP1 and H3K9me2,as opposed to no response in young cells;The senescent cells transfected with siPEA-15 showed significant increase of cell proliferation in 2 days as compared with the siControl transfected cells,and the difference was more significant in 4 days after the transfection." PERK1/2 -- Knockdown//Immunostaining Knockdown of PEA-15 by siPEA-15 transfection significantly induced pErk1/2 translocation. -- -- -- -- Human L cellular senescence 25725291 Sen_G_0489 FGF2 2247 protein coding B-MSC Bone marrow Aging Prevent SA-β-gal activity assay "β-Gal-positive cells were highly observed in control cells and those treated with EGF and HGF, but senescent cells were detected only rarely in FGF-2-treated groups after 2 months of culture." -- -- -- -- -- -- -- -- Human L cellular senescence 24491556 Sen_G_0490 FGF4 2249 protein coding B-MSC Bone marrow Aging Prevent SA-β-gal activity assay "β-Gal-positive cells were highly observed in control cells and those treated with EGF and HGF, but senescent cells were detected only rarely in FGF-4-treated groups after 2 months of culture." -- -- -- -- -- -- -- -- Human L cellular senescence 24491556 Sen_G_0491 SETD1A 9739 protein coding MDA-MB-231 -- Aging Prevent Cell proliferation assay//SA-β-gal activity assay//Knockdown "Remarkably, knockdown of SETD1A(SETD1A-KD) suppresses proliferation and triggers prompt (72h) and massive cellular senescence, with very large cells expressing characteristic ?-galactosidase (?-gal) activity." SKP2 -- Western blot//qPCR//SA-β-gal activity assay We first confirmed that endogenous SKP2 mRNA and protein are indeed suppressed in MDA-MB-231 cells following SETD1A-KD:taining for ?-Galactosidase shows that induction of SKP2 partially suppressed the emergence of ?-Gal-positive senescent cells by 50% following SETD1A-KD. -- -- -- -- Human HL cellular senescence 31253781 Sen_G_0492 SKP2 6502 protein coding "MDA-MB-231,BT-549" -- Aging Prevent SA-β-gal activity assay Staining for β-Galactosidase shows that induction of SKP2 partially suppressed the emergence of ?-Gal-positive senescent cells by 50% following SETD1A-KD. p21//p27 Downregulation//Downregulation Western blot Inducible expression of SKP2 in SETD1A-KD cells(using two different shSET1A constructs) effectively reduced the expression of p27 and p21 proteins. -- -- -- -- Human HL cellular senescence 31253781 Sen_G_0493 CXCR2 3579 protein coding "MEF,IMR-90" Embryo Aging Accelerate SA-β-gal activity assay//BrdU assay//Immunostaining//Growth curve assay "When viruses expressing CXCR2 were used to infect IMR-90 cells,both caused retarded growth culminating in premature senescence.The proportion of cells staining positively for senescence-associated β-ga-lactosidase (β-Gal) activity and displaying senescence-associated heterochromatin foci was elevated in cultures transduced with CXCR1 compared with controls.The cells showed reduced incorporation of BrdU and displayed characteristic features of senescence." -- -- -- -- p53//Rb --//-- Crystal violet staining "To this end we analyzed the effects of CXCR2 in WI-38 human fibroblasts in which p53 or pRb functions were disrupted using the HPV E6 and E7 proteins, respectively. The data confirm the importance of p53 but imply that the Rb pathway is also involved in establishing the CXCR2-induced arrest in human cells." Human L cellular senescence 18555777 Sen_G_0494 NSUN2 54888 protein coding 2BS -- Aging Prevent SA-β-gal activity assay//Knockdown "Knockdown of NSun2 inhibited cell growth and increased the proportions of cells displaying the protein marker senescence-associated (SA)-β-galactosidase (~37.5% vs. ~91.0%), while overexpression of NSun2 promoted cell growth and reduced SA-β-galactosidase activity (~33.5% vs. ~10.6%)." p27//CDK1//CDC25C Downregulation//Upregulation//Upregulation Western blot "Infection with the NSun2-expressing lentiviruses reduced the levels of p27 by ~70%, while infection with NSun2 shRNA-expressing lentiviruses increased the levels of p27 by ~5.1 fold.In contrast, the levels of proteins CDK1 and CDC25C increased in cells with overexpressed NSun2 (by ~2.8 fold for CDK1; by ~1.5 fold for CDC25C) but decreased in cells with silenced NSun2 (by ~70% for both CDK1 and CDC25C)." -- -- -- -- Human HL cellular senescence 26687548 Sen_G_0495 CXCL1 2919 protein coding "NOF,OSCC cell" Mucosa Oral cancer Accelerate SA-β-gal activity assay "Upon treatment with CXCL1 recombinant proteins, a significantly increased percentage of SA-β-Gal-positive cells was detected in treated cells compared with non-treated NOFs (p < 0.05)." -- -- -- -- -- -- -- -- Human L cellular senescence 29360827 Sen_G_0496 IL6 3569 protein coding "NOF,OSCC cell" Mucosa Oral cancer Accelerate SA-β-gal activity assay "Upon treatment with IL-6 recombinant proteins, a significantly increased percentage of SA-β-Gal-positive cells was detected in treated cells compared with non-treated NOFs (p < 0.05)." -- -- -- -- -- -- -- -- Human L cellular senescence 29360827 Sen_G_0497 PIK3CA 5290 protein coding EC Umbilical cord Vascular malformations Accelerate Cell morphological analysis//SA-β-gal activity assay The expression of Activate PI3K evidently modified EC morphology by dramatically increasing average cell size;Indeed the expression of Activate PI3K increased the amount of β-galactosidase positive cells. -- -- -- -- -- -- -- -- Human HL cellular senescence 29352118 Sen_G_0498 CCN1 3491 protein coding "A549,NCI-H460,NCI-H520" -- Lung cancer Accelerate Flow cytometry//Cell morphological analysis//SA-β-gal activity assay//BrdU assay "The CCN1-treated H460 cells showed prominent growth retardation starting at passage number 8. For A549 and H520 cells,significant growth suppression was also observed starting at passage number 11 and 13,respectively；Analysis by flow cytometry for cell cycle distributions of H460, A549, and H520 cells at passage number 11, 14, and 16, respectively, indicated that CCN1 induced cell cycle arrest in the G1 phase in all three NSCLC cell lines. The CCN1-treated cells exhibited a flattened morphology with concomitant cell enlargement, the presence of vacuole-rich cytoplasm, and high expression of perinuclear senescence-associated-β-galactosidase.Addition of CCN1 reduced BrdU incorporation to ~20 % of that observed in control samples with no or mock treatment in all three cell lines, suggesting the cessation of DNA synthesis in the senescence-like cells." -- -- -- -- p53-p21 Upregulation Cell morphological analysis//SA-β-gal activity assay//Knockdown "Addition of CCN1 to the p21-deficient cells failed to suppress cell growth, to induce senescence-like morphologies (6C), or to enhance SA-β-Gal activities (6D). Similar results were found in the p53-knockdown cells." Human L cellular senescence 23553737 Sen_G_0499 DUSP4 1846 protein coding WI-38 -- Aging Accelerate Cell morphological analysis//SA-β-gal activity assay//MTT assay "In contrast to the vigorously proliferating, spindle-like control fibroblasts, MKP2 cells exhibit the distinct flattened, enlarged morphology of senescent cells, sensitivity to contact inhibition, and loss of replicative capacity.we found that throughout their lifespan, the percentage of stained cells was always higher in the cultures expressing MKP2." -- -- -- -- ERK Downregulation Knockdown "MKP2 expression resulted in a dramatic decrease of thelifespan of the ERK2:NLS line while it had no effect in the lifespan of the phosphatase resistant ERK2(D319N):NLS cells. Thus, we concluded that MKP2 overexpression inhibits proliferation and accelerates senescence by inhibiting nuclear ERK signaling and not by inactivating other MAPK family members." Human L cellular senescence 17145763 Sen_G_0500 RAC1 5879 protein coding HBMEC -- Aging Prevent SA-β-gal activity assay "Quantification of β-galactosidase positive HBMECs showed a significant increase in β-galactosidase staining following Rac 1 inhibition,suggesting that Rac 1 inhibition plays a critical role in senescence induction in the HBMECs." -- -- -- -- -- -- -- -- Human L cellular senescence 31513781 Sen_G_0501 FLT1 2321 protein coding HBMEC -- Aging Accelerate SA-β-gal activity assay//Knockdown We then performed siRNA mediated VEGFR-1 knockdown as before and confirmed that the siRNA mediated VEGFR-1 knockdown reduced senescence in the Aβ1–42 oligomertreated HBMECs as measured by β-galactosidase staining. -- -- -- -- -- -- -- -- Human L cellular senescence 31513781 Sen_G_0502 JUN 3725 protein coding MCF-7 -- Aging Prevent SA-β-gal activity assay "However, for MCF7-c-Jun cells maintained in the absence of doxycycline to induce c-Jun expression, the percentage of cells staining positive for β-galactosidase activity was much lower and was not significantly increased after vinblastine treatment." -- -- -- -- -- -- -- -- Human L cellular senescence 17126817 Sen_G_0503 CXCL8 3576 protein coding PMSC -- Aging Prevent Cell morphological analysis//SA-β-gal activity assay//Flow cytometry//CCK-8 assay "The enlarged and flattened PMSCs stained blue, indicating they have become senescent. The percentage of SA-β-gal-positive PMSCs was significantly increased on day9 after IL-8 knockdown.A flow cytometric analysis indicated that the cellular G2/M phase ratio was significantly increased, indicating that the G2-M phase was prolonged in the IL-8-silenced PMSCs when compared with their controls.The IL-8-silenced PMSCs showed significant growth retardation." -- -- -- -- Akt-FOXO3a//CXCR2 --//-- qRT-PCR//Western blot "Increased p-AKT expression and decreased FOXO3a protein expression in IL-8-silenced PMSCs were verified by western blot analysis.We found that in IL-8-silenced PMSCs, the expression levels of CXCR2 ligands were time-dependent: most of the CXCR2 ligands were present at reduced levels in pre-senescent shIL-8 PMSCs,while their expression later increased along with the cellular senescence process." Human L cellular senescence 28418782 Sen_G_0504 PRKDC 5591 protein coding Fibroblast -- Idiopathic Pulmonary Fibrosis Accelerate qRT-PCR "Transcriptomic analysis of Nu7441(DNA-PKcs inhibitor)-treated and untreated fibroblasts showed that this treatment caused a significant increase in the senescence-associated CDKN1A, CDKN1B, CDKN2A, NOX4." -- -- -- -- -- -- -- -- Human L cellular senescence 31464599 Sen_G_0505 TNF 7124 protein coding ECFC -- Aging Prevent SA-β-gal activity assay We incubated ECFCs with either recombinant TACE or an anti-TNFR2 neutralizing antibody for 6 days and determined development of premature senescence by staining for senescence-associated β-galactosidase. p38 Activation SA-β-gal activity assay//Colony formation assay We examined the effect of p38 inhibition on the expression of tmTNF and found that blocking p38 completely prevented the loss of tmTNF and subsequent development of premature senescence. tmTNF-TNFR2 -- Western blot "We treated ECFCs with either TACE or anti-TNFR2 neutralizing antibody and detected the presence of p16ink, a senescence-associated cell cycle regulating protein by Western blot and found that p16ink levels increased dramatically during the course of the 6-day treatment, further confirming that loss of the tmTNF/TNFR2 signaling axis results in premature senescence of ECFCs." Human L cellular senescence 27076598 Sen_G_0506 EGF 1950 protein coding "SK-ES-1,RD-ES" -- Ewing sarcoma Prevent Colony formation assay//Cell counting These assays showed that EGF significantly increased both the proliferation rate and survival in SK-ES-1 and RD-ES cells;Evidence of senescence induction was evaluated with a colorimetric assay after exposure of ES cells to AG1478. The percentage of senescent cells was increased in cells treated with any drug dose compared to controls. -- -- -- -- -- -- -- -- Human L cellular senescence 29539615 Sen_G_0507 SOX1 6656 protein coding Hep3B -- Hepatocellular carcinoma Accelerate Flow cytometry//SA-β-gal activity assay//Western blot "Our data showed that ectopic expression of SOX1 increased number of cells in G1 while decreasing number of cells in S phase in Hep3B cells.In Hep3B cells,SOX1 expression significantly enhanced the protein level of p21 and p27 but suppressed expression of CDK4 and CDK6 as compared with the control cells.We found that expression of SOX1 in Hep3B cells could enhance the signal of SA-β-gal staining." p21//p27 Upregulation//Upregulation Western blot//Flow cytometry "In Hep3B cells, SOX1 expression significantly enhanced the protein level of p21 and p27 but suppressed expression of CDK4 and CDK6 as compared with the control cells. In the SOX1-expressing HepG2 cells, p21 and p27 were also dramatically upregulated. However, there, was no significant difference in the protein levels of CDK4 and CDK6." -- -- -- -- Human HL cellular senescence 22767186 Sen_G_0508 SPAG9 9043 protein coding MDA-MB-231 -- Breast cancer Prevent Cell cycle analysis//Knockdown//Western blot//SA-β-gal activity assay "Knockdown of SPAG9 by SPAG9 shRNA1(67.03 %) and shRNA2 (65.14 %) resulted in accumulation of most of the cells in G1 phase as compared to NC shRNA (63.50 %) transfected cells. Also, the percentage of G2-M phase cells showed decrease in SPAG9 shRNA [shRNA1 (23.08 %), shRNA2 (26.42 %)]-transfected cells as compared to NC shRNA (28.26 %)-transfected cells.The result showed that there was a significant decrease in cyclins and cyclin-dependent kinases such as cyclin B1, cyclin D1, cyclin E,CDK1, CDK4, and CDK6. Also, upregulation was observed in case of tumor suppressor protein, p21. The percentage of senescent cells was significantly higher(p < 0.0001), 52.6 % and 67.0 %, when transfected with SPAG9 shRNA1 and shRNA2, respectively, as compared to 7.4 % when transfected with NC shRNA." p21//Cyclin B1//Cyclin D1//Cyclin E//CDK4//CDK6 --//--//--//--//--//-- Immunostaining//Western blot "IHC analysis of tumor serial sections revealed significantly enhanced immunoreactivity of p21 and decreased immunoreactivity of cyclin B1, cyclin D1,cyclin E, CDK4, and CDK6 in SPAG9 shRNA2-treated mice as compared NC shRNA-treated mice.Western blotting was carried out to study the various molecules in different phases of cell cycle, which showed that there was a significant decrease in cyclins and cyclindependent kinases such as cyclin B1, cyclin D1, cyclin E,CDK1, CDK4, and CDK6. Also, upregulation was observed in case of tumor suppressor protein, p21." -- -- -- -- Human L cellular senescence 27449044 Sen_G_0509 GFPT2 9945 protein coding HBEC3-KT -- Aging Prevent SA-β-gal activity assay//Knockdown Genetic knockdown of GFPT2 and UAP1 in NSCLC cells revealed increased markers of senescence and reduced clonogenic potential. -- -- -- -- -- -- -- -- Human HL cellular senescence 30130254 Sen_G_0510 UAP1 6675 protein coding HBEC3-KT -- Aging Prevent SA-β-gal activity assay//Knockdown Genetic knockdown of GFPT2 and UAP1 in NSCLC cells revealed increased markers of senescence and reduced clonogenic potential. -- -- -- -- -- -- -- -- Human HL cellular senescence 30130254 Sen_G_0511 TP53 7157 protein coding HCA2 -- Aging Accelerate Western blot//SA-β-gal activity assay//FACS analysis "Transient expression of p53 in cells released to G2 phase for 4 hr from a thymidine block resulted in loss of mitotic regulators, cessation of cell proliferation, and an increase in the SA-β-gal-positive fraction." -- -- -- -- -- -- -- -- Human L cellular senescence 24910096 Sen_G_0512 RB1 5925 protein coding HCA2 -- Aging Accelerate Western blot//SA-β-gal activity assay//FACS analysis "Transient expression (24 hr) of pRb7LP by the addition of doxycycline to G2 cells, in the presence of a Cdk2 inhibitor to prematurely activate Cdh1, resulted in impaired cell proliferation and an increase in the population of cells staining positive for SA-β gal.The resulting senescence phenotype of the cells was further confirmed by the induction of p16 and senescence-associated cytokines such as IL-6 and IL-8." -- -- -- -- -- -- -- -- Human L cellular senescence 24910096 Sen_G_0513 TXNRD1 7296 protein coding NIH-3T3 -- Aging Prevent Western blot//SA-β-gal activity assay "We show that expression of F-A-TrxR1-HA inhibited SIPS in fibroblasts, as shown by the lesser number of senescence-associated β-galactosidase-positive cells after oxidative stress as compared with wt-TrxR1-HA-expressing cells.We found that expression of the constitutively Activate F-A-TrxR1-HA inhibited ROS-induced upregulation of p53, expression of p21Waf1/Cip1protein and activation of a p53 responsive element 24 h after oxidative stress,as compared with wt-TrxR1-HA." Caveolin-1 Downregulation Western blot "We found that caveolin 1 inhibits TrxR1 activity by preventing the formation of TrxR1 homodimers, as shown by increased levels of dimeric TrxR1 in cells in which endogenous caveolin 1 expression was reduced by short interfering RNA." -- -- -- -- Human L cellular senescence 19820694 Sen_G_0514 CDKN2A 1029 protein coding MCF-7 -- Aging Accelerate Cell morphological analysis//Flow cytometry "Microscopic analysis revealed that control cells formed a stably proliferating population, whereas cells transfected with the cdk inhibitors, p16 stopped proliferation and adopted a senescent morphology. Flow cytometry data confirmed proliferation of control cells and induction of a G1-phase arrest in cells expressing exogenous p16." -- -- -- -- -- -- -- -- Human L cellular senescence 19648966 Sen_G_0515 CDKN1A 1026 protein coding MCF-7 -- Aging Accelerate Cell morphological analysis//Flow cytometry "Microscopic analysis revealed that control cells formed a stably proliferating population, whereas cells transfected with the cdk inhibitors, p21 stopped proliferation and adopted a senescent morphology. Flow cytometry data confirmed proliferation of control cells and induction of a G1-phase arrest in cells expressing exogenous p21." -- -- -- -- -- -- -- -- Human L cellular senescence 19648966 Sen_G_0516 CCN1 3491 protein coding HSC Liver Liver disease Accelerate Immunostaining//SA-β-gal activity assay "We stained liver sections for senescence-associated markers, Ki67 and SA-β-gal. Immunohistochemistry for Ki67 revealed that Mdr2-/- livers contained nearly 2-fold more proliferating cells compared with control and Dko mice." -- -- -- -- -- -- -- -- Human HL cellular senescence 29105104 Sen_G_0517 EIF4EBP1 1978 protein coding "A549,AD32" -- Aging Prevent SA-β-gal activity assay "AD32 cells had a higher basal level of senescence-associated β-galactosidase (SA-β-gal)–positive cells than A549—0.40% and 0.07%, respectively.（AD32 cells have decreased levels of 4E-BP1 mRNA and protein, relative to the parental discodermolide-sensitive A549 cells）." p53 Downregulation Western blot "AD324E-BP1cells had decreased levels of p53 protein, indicating that p53 levels may be decreased by overexpression of 4E-BP1 in AD32 cells." -- -- -- -- Human L cellular senescence 21173253 Sen_G_0518 TP73 7161 protein coding HCT116 -- Aging Prevent SA-β-gal activity assay//Flow cytometry//Knockdown "The senescence-associated β-galactosidase (SA-β-gal) staining showed that p73 knockdown significantly increased the proportion of positive cells (~89.1% in p73i-1 and 79.7% in p73i-2 transfected cells) at 5 dpi, compared to the irradiated controls (~41.2%).However, the proportion of cells in the G2 phase increased significantly in irradiated p73-knockdown cells (30.2% in p73i-1 and 27.2% in p73i-2 transfected cells),compared to that in irradiated control cells (17.8%)." Δ133p53 Binding Western blot//Co-IP "The western blot revealed that Δ133p53,but not full-length p53,co-immunoprecipitated with p73 at 12 hpi." -- -- -- -- Human HL cellular senescence 29511339 Sen_G_0519 ERBB2 2064 protein coding MCF 10A -- Cancer Accelerate SA-β-gal activity assay//Cell morphological analysis//Growth curve assay "We expressed HER2 in HSF1-depleted cells, a significant growth inhibition was observed, associated with a significant change in cells’ appearance, including enlarged, flattened morphology and extensive vacuolization reminiscent of senescence.HER2 expression in control MCF-10A cells led to a significant increase in β-gal-positive population (B30%)." p21//Survivin Upregulation//Downregulation Western blot//Co-IP "P21 was mildly upregulated by HER2 expression in control cells.Upon HER2 expression, survivin levels were dramatically decreased in shHSF1 cells." -- -- -- -- Human L cellular senescence 20622894 Sen_G_0520 HSF1 3297 protein coding MCF 10A -- Cancer Prevent SA-β-gal activity assay "HER2 expression in control MCF-10A cells led to a significant increase in β-gal-positive population (B30%). Importantly, expression of HER2 in shHSF1 MCF10A cells resulted in about 70% of β-gal-positive cells." -- -- -- -- -- -- -- -- Human L cellular senescence 20622894 Sen_G_0521 PTEN 5728 protein coding BJ-T -- Aging Prevent SA-β-gal activity assay//Cell morphological analysis Cells expressing PTEN shRNA exhibited a significant increase in cell size and senescence associated β-galactosidase activity (SAβGAL). -- -- -- -- PI3K-Akt Activation Western blot Cells with depleted PTEN protein levels or exhibited increased levels of phospho-AKT and phosphorylation of the AKT substrate PRAS40. Human L cellular senescence 21909130 Sen_G_0522 PIK3CA 5290 protein coding BJ-T -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis Cells expressing PIK3CAE545K exhibited a significant increase in cell size and senescence associated β-galactosidase activity (SAβGAL). -- -- -- -- PI3K-Akt Activation Western blot Cells with expressing PIK3CAE545K exhibited increased levels of phospho-AKT and phosphorylation of the AKT substrate PRAS40. Human L cellular senescence 21909130 Sen_G_0523 AKT1 207 protein coding BJ-T -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis//qRT-PCR "Cells expressing activated AKT showed a significant increase in SAβGAL and cell size.AKT-induced senescence is characterised by an SASP, with increased secretion of IL-1a, IL-1b, IL-6 and IL-8." -- -- -- -- p53//mTORC1 Upregulation//-- Knockdown//SA-β-gal activity assay//Western blot "SAβGAL positivity was significantly reduced in BJ-T-myr-AKT/p53 stable knockdown cells as compared with control BJ-T-myr-AKT cells. Acute p53 knockdown, confirmed by immunoblot analysis, rescued the reduced proliferation of myr-AKT1-expressing cells to that of control cells .Upon treatment with rapamycin,the percentage of AKT cells positive for SAβGAL was significantly reduced .Rapamycin treatment also dramatically reduced AKT-induced effects on cell size, and the SASP, indicating that mTORC1 activity is critical for PI3K/AKT-driven senescence." Human L cellular senescence 21909130 Sen_G_0524 HRAS 3265 protein coding BJ-T -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis//SAHF Cells expressing activated AKT isoforms and H-RASV12showed a significant increase in SAβGAL and cell size.The robust induction of SAHF formationwith was observed when RAS was activated. -- -- -- -- -- -- -- -- Human L cellular senescence 21909130 Sen_G_0525 PIK3CA 5290 protein coding EC -- Vascular malformations Accelerate SA-β-gal activity assay//Cell morphological analysis//EdU assay "The expression of Activate PI3K evidently modified EC morphology by dramatically increasing average cell size.Indeed the expression of Activate PI3K, both H1047R and E545K mutants, increased the amount of β-galactosidase positive cells.We measured DNA replication rates by means of EdU incorporation assay. EC-H1047R and EC-E545K showed higher DNA replication rates, which were particularly elevated when EC were stimulated with VEGF-A." -- -- -- -- -- -- -- -- Human L cellular senescence 29352118 Sen_G_0526 CDK4 1019 protein coding IMR-90 -- Aging Prevent Cell proliferation assay We found that these kinases efficiently blocked PML-induced growth arrest and senescence in normal cells. -- -- -- -- -- -- -- -- Human HL cellular senescence 27206849 Sen_G_0527 CDK6 1021 protein coding IMR-90 -- Aging Prevent Cell proliferation assay We found that these kinases efficiently blocked PML-induced growth arrest and senescence in normal cells. -- -- -- -- -- -- -- -- Human HL cellular senescence 27206849 Sen_G_0528 SIRT6 51548 protein coding Primary human keratinocyte -- Aging Prevent Knockdown//SA-β-gal activity assay//Western blot "SIRT6 knockdown significantly increased senescence of primary keratinocytes,and this effect was reversed by depletion of RELA by RNAi." H3K9 -- CHIP//Western blot "ChIP analysis revealed that H3K9 acetylation is induced following TNF-a treatment in SIRT6-proficient control cells at the promoters of multiple NF-kB target genes, consistent with transcriptional induction. In cells depleted of SIRT6, H3K9 was hyperacetylated at these promoters in response to TNF-a." NF-κB Downregulation Luciferase reporter assay//Knockdown "SIRT6 depletion led to constitutive NF-kB reporter gene activity, which, upon TNF-a treatment,was further enhanced to levels considerably higher than in SIRT6-proficient control cells ." Human HL cellular senescence 19135889 Sen_G_0529 PNPT1 87178 protein coding HeLa -- Aging Accelerate Flow cytometry//Western blot Overexpression of hPNPaseold-35 induces a senescence-like growth arrest and also generates ROS. -- -- -- -- NF-κB Activation Western blot//RT-PCR//ELISA//Luciferase reporter assay "However,infection with Ad.hPNPaseold -35 resulted in a 10- to 12-fold induction in relative luciferase activity in comparison with control or Ad.vec-infected cells.On Ad.hPNPaseold-35 infection, the binding pattern changed, with the p50/p50 homodimer disappearing, and the binding of the p50/p65 heterodimer increasing markedly.Expressions of mRNAs and secreted proteins of IL-6 and IL-8, two NF-kB target genes, were analyzed by RT-PCR and ELISA, respectively,after Ad.hPNPaseold-35infection." Human HL delay aging 15492272 Sen_G_0530 BLVRA 644 protein coding HDF -- Aging Prevent Cell morphological analysis//SA-β-gal activity assay//Flow cytometry//Western blot//Knockdown "Morphological analysis indicated that HDF cells became enlarged and flattened after BLVRA shRNA treatment.Moreover,knockdown of BLVRA led to induce the expression of the senescence marker SA-β-gal.BLVRA knockdown cells were arrested in the G0-G1 phase of the cell cycle to approximately 78% of the cells whereas 53% of random shRNA-treated cells were arrested in the G0-G1 phase of the cell cycle.Transfection of the cells with shRNA- BLVRA induced the expres-sion levels of p53, 16, and p21 significantly." -- -- -- -- -- -- -- -- Human L delay aging 21099244 Sen_G_0531 WRN 7486 protein coding WI-38 -- Werner syndrome Prevent Cell morphological analysis//SA-β-gal activity assay//Flow cytometry//BrdU assay "We found that upon withdrawal of WRN, the morphology of untransformed primary fibroblasts changed progressively within 5 days posttransfection. The cells became enlarged, flattened and developed the SA-β-Gal activity with a concomitant moderate decline of cumulative cell number.The significant hypophosphorylation of Rb was accompanied by decreased proliferation of the WRN-deficient cells, as evidenced by an average 40% reduction of bromodeoxyuridine incorporation (data not shown) and a similar decrease in S-phase cells with an increased proportion of G0-G1 cells by FACS analysis." p16 -- Knockdown//Western blot Untransformed fibroblasts responded to acute knockdown of WRN with a minimal elevation of the levels of the p16 cell-cycle inhibitors within the time frame of 5 days. p53//Rb --//-- Knockdown//Western blot "Untransformed fibroblasts responded to acute knockdown of WRN with a minimal elevation of the levels of p21,the downstream p53 target within the time frame of 5 days. However,an almost complete disappearance of retinoblastoma (Rb) phosphorylation and, to a lesser degree, the dephosphorylated Rb protein was evident ." Human HL delay aging 16287861 Sen_G_0532 BLM 641 protein coding WI-38 -- Werner syndrome Prevent Cell morphological analysis//SA-β-gal activity assay//Knockdown "RNAi directed against BLM resulted in a similar or somewhat greater reduction in cell number and appearance of SA-β-Gal activity, albeit with less evident flattening morphology of the cells." p16 -- Knockdown//Western blot Untransformed fibroblasts responded to acute knockdown of BLM with a minimal elevation of the levels of the p16 cell-cycle inhibitors within the time frame of 5 days. p53//Rb --//-- Knockdown//Western blot "Untransformed fibroblasts responded to acute knockdown of BLM with a minimal elevation of the levels of p21,the downstream p53 target within the time frame of 5 days .Acutely BLM-depleted cells also showed marked dephosphorylation of Rb protein." Human HL delay aging 16287861 Sen_G_0533 GCG 2641 protein coding HUVEC -- Diabetes Prevent SA-β-gal activity assay Treatment of HUVECs with GLP-1 attenuated the increase of senescent cells in a dose-responsive manner. DPP-4//CREB Upregulation//Activation Western blot "Treatment of the ZDF animals with vildagliptin resulted in a significant reduction of DPP-4 activity and an almost 6-fold increase of GLP-1 plasma levels.The vildagliptin treatment did, however, decrease cellular senescence in these animals, to levels almost comparable to those of lean rats (2.6±0.6% versus 2.3±0.5%). This suggests that increased GLP-1 levels by DPP-4 inhibition have a protective effect on the vasculature.Western blot analysis using an anti phosphorylated CREB antibody showed that GLP-1 treatment increased relative phosphorylated CREB levels by 52% compared with the control." cAMP-PKA -- Premature senescence assay "PKA inhibition by H89 was also sufficient to block the GLP-1-mediated protective effect on HUVECs. H89 inhibition showed a dose response effect on cellular senescence in combination with GLP-1, with a complete abolishment of the GLP-1 protective effect at a concentration of 1 umol/L H89. Similar results were obtained with another PKA inhibitor KT5720." Human L delay aging 20448207 Sen_G_0534 SRC 6714 protein coding Human endothelial cell -- Aging Accelerate SA-β-gal activity assay "Incubation with H2O2 significantly increased acidic β-galac-tosidase–positive cells. Interestingly, coincubation with the Src kinase inhibitor PP2 (500 nmol/L) completely blocked the induction of premature senescence indicating that Src kinase activation contributes to endothelial cell senescence." -- -- -- -- -- -- -- -- Human L delay aging 14963003 Sen_G_0535 SIRT1 23411 protein coding PAEC -- Aging Prevent SA-β-gal activity assay//Flow cytometry "Results from SA-β-gal staining, as well as flow cytometric analysis, suggested that SIRT1 alleviated cellular senescence." -- -- -- -- LKB1-AMPK//Akt Activation//Activation Western blot//SA-β-gal activity assay "Importantly, the amount of LKB1 and phosphoAMPK(T172) were significantly lower in SIRT1 transgenic mice. In addition, decreased SIRT1 expression and elevated LKB1/AMPK levels were also observed in the aorta tissues of old mice by comparing to those in young mice.SIRT1 and resveratrol treatment increased Akt(Ser473) phosphorylation in normal cultures,Inhibition of Akt by either Akt inhibitor or kinase-dead Akt could induce senescence only when the experiment was performed under serum containing conditions .These results largely mirrored the effects of Akt inhibition on AMPK activation." Human L delay aging 20203304 Sen_G_0536 BMI1 648 protein coding Renal cell Kidney Renal tubulointerstitial injury Prevent Immunostaining//TUNEL assay//SA-β-gal activity assay "Results showed that the percentage of Ki67-positive cells was decreased dramatically, whereas the percentage of TUNEL-positive cells, SA-β-gal positive areas, 8-OHdG-positive cells, CD3-positive and F4/80-positive inflammatory cells were significantly increased in Bmi-1-/-mice compared with WT mice." -- -- -- -- -- -- -- -- Human HL delay aging 28790310 Sen_G_0537 CDKN2A 1029 protein coding Renal cell Kidney Renal tubulointerstitial injury Accelerate Immunostaining//SA-β-gal activity assay//Knockdown//qRT-PCR//Western blot "P16 deletion was significantly rescued the abnormalities in renal cell proliferation, apoptosis and senescence, DNA damage and inflammatory cell infiltration observed in Bmi-1 -/- mice.However,they were reduced significantly in Bmi-1-/- p16 -/- mice compared with Bmi-1-/- mice.These results demonstrated that p16 deletion ameliorated the proinflammatory secretory phenotype caused by Bmi-1 deficiency." -- -- -- -- -- -- -- -- Human HL delay aging 28790310 Sen_G_0538 PLA2R1 22925 protein coding MRC-5 -- Hutchinson–Gilford progeria syndrome Accelerate qRT-PCR//SA-β-gal activity assay//Cell counting//Crystal violet assay//Knockdown "Constitutive expression of progerin resulted in proliferation arrest as judged by reduced number of cells observed using crystal violet staining and growth curves and reduced expression of the proliferation marker Ki67, and increased frequency of SA‐β‐Gal‐positive cells and increased expression of p21 (CDKN1A) and the SASP component IL‐8.Knockdown of PLA2R1 with two independent shRNAs abolished all these hallmarks of cellular senescence." -- -- -- -- p53-FDPS -- qRT-PCR//Immunostaining "As expected,progerin increased P‐ATM and γH2AX DNA damage marks, p53 phosphorylation, the p53 transcriptional target p21, and FDPS, and these inductions were abolished upon PLA2R1 knockdown." Human L delay aging 30216637 Sen_G_0539 CXCR4 7852 protein coding SH-SY5Y -- Alzheimer's disease Prevent CCK-8 assay//MTT assay//Knockdown "Moreover, the data of CCK8 assay demonstrated that the cell activity was decreased in siCXCR4 cells. Meanwhile,siCXCR4 cells have significant deficient effects on cell proliferation and activity compared with normal and control groups." Akt//CREB//p53 Activation//Activation//Activation Western blot//Immunostaining//Knockdown "Using Western Blotting assay, we observed that the phosphorylation at 308 of AKT in siCXCR4 group was robustly inhibited.Using a pixelby-pixel colocalization analysis, the results illustrated that fluorescent signals of AKT were clearly merged with CXCR4 on plasma membrane. Meanwhile, the phosphorylation of CREB was significantly decreased, while the level of P53 was increased in CXCR4 knock down cells compared with normal or control groups." CXCL12-CXCR4 Activation Immunostaining We firstly found a strong colocalization of CXCR4 and AKT on plasma membrane stimulating by 100ng/ml CXCL12. Human L delay aging 30080220 Sen_G_0540 TP53 7157 protein coding HSC "Spine,tibias,Skin " Aging Accelerate Histological staining//Flow cytometry//BrdU assay "P72 mice showed a delayed development of all these aging-related changes compared with R72 mice, with the most obvious differences observed at the age of 18 months.R72 mice showed more significant decreases in both skin dermal thickness and subcutaneous adipose thickness compared with P72 mice.R72 mice showed a more rapid increase in the numbers of LT-HSCs than P72 mice during aging.While there was no significant difference in LT-HSC numbers between young 129SVslR72 and P72 mice, much higher LT-HSC numbers were observed in R72 mice than P72 mice at the age of both 12 and 18 months.The decrease of proliferation HSCs was more rapid in R72 mice than P72 mice during aging .R72 mice displayed a more obvious sign of osteoporosis than P72 mice.However, 12- and 18-month-old R72 mice showed a more pronounced decrease in the wound healing ability than age-matched P72 mice.Notably, 18-month-old R72 mice developed more pronounced lordokyphosis compared with age-matched P72 mice." p21 Upregulation RT-PCR//Western blot p21 mRNA expression levels in the bone marrow were slightly higher in P72 mice compared with R72 mice as determined by real-time PCR assays with this difference being more obvious in older mice than young mice. This difference in p21 expression levels was confirmed at the protein level as determined by Western-blot assays. -- -- -- -- Human L delay aging 29557783 Sen_G_0541 COL17A1 1308 protein coding HFSC Hair Aging Prevent Histological images analysis We generated HFSC-specificCol17a1-deficient mice (Col17a1cKO). We found that those mice also show thinning hair and graying hair.The heat map for the global transcriptome of activated HFSC fractions (aHFSCs) revealed a significantly close relationship between Col17a1cKO HFSCs and aged HFSCs. Most of those mice showed significantly fewer miniaturized HFs and an apparent retardation of hair loss even in mice surving for 24 months (N = 3 mice) and 32 months (N = 2 mice). ELANE -- Western blot "Indeed, primary keratinocytes treated with ELANE showed that both the 180-kD COL17A1 and its shed form of the 120 kD ectodomain are quickly degraded by ELANE in vitro." -- -- -- -- Human HL delay aging 26912707 Sen_G_0542 CDKN2A 1029 protein coding HDF -- Aging Accelerate Cell morphological analysis//qRT-PCR//Knockdown "This study showed that senescent HDFs transfected with p16INK4asiRNA showed changes of morphology from senescent morphology to morphology of young cells with the presence of small and spindle-shaped fibroblasts.The data showed that, in senescent HDFs, p16INK4amRNA was significantly upregulated (p < 0.05) compared to young HDF cells." Cyclin D1 -- Western blot "Cells transfected with p16INK4a siRNA showed downregulation (p < 0.05) of cyclin D1 compared to untreated senescent cells.In senescent cells, cyclin D1 was upregulated significantly (p < 0.05) when compared to young cells." -- -- -- -- Human L delay aging 27743340 Sen_G_0543 SIRT6 51548 protein coding U2OS -- Aging Prevent SA-β-gal activity assay "We detected significantly increased numbers of senescent U2OS cells (over ~20-fold higher than control cells) within a week of SIRT6 depletion by lentiviral shRNAs, and increased senescence was observed as early as 72 hours after transient transfection of SIRT6 siRNAs." H3K18Ac -- Western blot "Upon extending this analysis to new peptides, we found that SIRT6 robustly deacetylated lysine K18 on histone H3 peptides (H3K18Ac).SIRT6 also promoted H3K18Ac deacetylation when over-expressed in cells, while the mutant SIRT6 H133Y protein did not." -- -- -- -- Human HL delay aging 27043296 Sen_G_0544 ITGB3 3690 protein coding BF -- Aging Accelerate BrdU assay//SA-β-gal activity assay//Immunostaining//qRT-PCR "Indeed, expression of a retroviral vector encoding ITGB3 in BFs reduces their proliferation rate, quantified by measuring the percentage of cells incorporating bromodeoxyuridine (BrdU).Consistent with the activation of senescence, ITGB3 expression led to an increase in the number of cells staining positive for senescence-associated β-galactosidase (SA-β-Gal) activity, an accumulation of reActivate oxygen species (ROS), and a mild increase in the mRNA levels of different SASPs." CBX7 -- Western blot//Immunostaining//Knockdown//CHIP "Importantly,we observed that shCBX7 increases the number of cells presenting avβ3-stained FA complexes by IF. The regulation of β3 protein levels by CBX7 was also confirmed in IMR-90 fibroblasts.We checked the levels of the integrin heterodimer avb3 by immunofluorescence (IF)and the β3 subunit by immunoblot upon CBX7 knockdown or Cbx7 ectopic expression.Our data show a reduced binding of CBX7 to the ITGB3 TSS during OIS, suggesting that the endogenous upregulation of ITGB3 during OIS is due to the transcriptional deregulation of the locus by the loss of CBX7 binding." p53-p21//TGF-β Activation//Activation SA-β-gal activity assay//qRT-PCR "Using a previously characterized shRNA targeting TP53 (shp53) , we impaired not only the proliferation arrest induced by ITGB3 but also the increase in SA-β-Gal activity. The use of a short interfering RNA (siRNA) targeting TP53 (sip53) also impaired the growth arrest induced by ITGB3 expression. Our data demonstrate that both siRNAs against TGFBR2 overcome senescence induced by the overexpression of ITGB3, as shown by measuring the relative cell number and p21CIPlevels by IF. Indeed, qPCR analyses of a range of regulators implicated in the TGF-β pathway are upregulated in BFs expressing ITGB3." Human L delay aging 28273461 Sen_G_0545 DLX3 1747 protein coding B-MSC -- Aging Accelerate SA-β-gal activity assay//qRT-PCR//Western blot "The number of SA-β -gal+ staining cells 38% in TDO-BMSCs and 55% in CON-BMSCs suggested that BMSCs with DLX3 mutation remained a younger status while the normal BMSCs entered a premature senescence.After 72 h osteoinduction, aging-related markers p16INK4a and p15INK4b mRNA expression detected by real-time PCR and p16INK4a and GLB1 protein expression examined by western blot were significantly increased in WT-DLX3 but decreased in MT-DLX3 and TR-DLX3 when compared with EGFP-EV . Stemness markers Oct4 and Nanog mRNA expression detected by real-time PCR were significantly decreased in WT-DLX3 but increased in MT-DLX3 and TR-DLX3 when compared with EGFP-EV .The results showed that percentage of SA-β -gal-positive cells was significantly higher in WT-DLX3, but much lower in MT-DLX3 and TR-DLX3." -- -- -- -- -- -- -- -- Human HL delay aging 27924851 Sen_G_0546 CIZ1 25792 protein coding MEF Brain Neurodegenerative disease Prevent Flow cytometry//Comet assay//Behavioral assessment//Immunostaining "Using flow cytometry, MEFs from null mice showed cell-cycle defects and increased apoptosis (two-tailed t(8) = 3.14, P = 0.014) in comparsion with MEFs from WT littermates. For this purpose, we investigated DNA damage in the cerebellum and hippocampus of aged WT and null mice with comet assays and immunohistochemistry. We found increased % tail DNA in the cerebellum (two-tailed t(10) = 12.14, P <0.0001) and hippocampus (two-tailed t(10) = 14.07,P <0.0001) of aged Ciz1?/ ? mice as compared with WT littermates .Behaviorally, male and female Ciz1?/ ? mice were more aggressive than their WT littermates in the dominance tube (P < 0.0001). Similar to motor and behavioral findings, we also observed cognitive dysfunction in Ciz1?/ ? mice as assessed by cross maze and Morris water maze testing." -- -- -- -- NF-κB -- Western blot "Consistent with our gene expression data, we observed increased protein levels of NF-κB (p65) in the nuclear extracts from the cerebellae and hippocampi." Human L delay aging 29154038 Sen_G_0547 JAG1 182 protein coding MSC -- Aging Prevent SA-β-gal activity assay//Flow cytometry "As we expected,activation of Notch signaling by JAG1 led to a reduced cell senescence in both 1- and 5-day sheet cultures, as demonstrated by decreased frequencies of SA-β-gal positive cells (8.25±1.5% and 20.5±4.2%, respectively) when compared with MSCs from lgG control groups .By comparison, the G0 phase cells in JAG1 groups was 45.2±2.8% and 57.5±8.2% in the 1-day and 5-day cultures respectively, and significantly lower than those in the lgG group." p16//CCND1 Downregulation//Upregulation RT-PCR//Western blot "In lgG control group,relative quantification by RT -PCR revealed an ~ 5-fold up-regulation of p16 RNA and a 2.7-fold up-regulation of p21 RNA in 5-day cultures compared with 1-day cultures, which suggests that these two factors act synergistically to induce MSC senescence. Finally, protein expression of p16 in western blot confirmed our PCR results by showing thinner band in JAG1 treated cells and thicker band in Hes1 deficient cells, and no significant change was observed in protein expression of p21 .Consistent with our previous finding, CCND1 expression was induced by JAG1 in 1-day cultures and no significant changes were observed in 5-day cultures compared with lgG controls." JAG1-Notch-Hes1 Activation RT-PCR//Western blot//SA-β-gal activity assay "Therefore, we further measured Notch target Hes1 expression in 1- and 5-day sheet cultures. RT -PCR analysis of total RNA revealed a decreased expression of Hes1from 1-day to 5-day cultures in control lgG-coated plates, while Hes1 expression at both time points was significantly increased by JAG1.Western blot analysis using protein from 5-day sheet cultures further confirmed these knockdown results by showing a significantly reduced Hes1 protein expression in shRNA lentivirus-infected MSCs with or without JAG1 treatment.More importantly, although the cellular senescence in 5-day cultures was significantly inhibited by JAG1-mediated Notch activ ation, this inhibitory effect was almost completely abrogated after knocking down Hes1 expression in these MSCs." Human L delay aging 28151468 Sen_G_0548 CD9 928 protein coding A549 "Bone,Muscle,Adipose,Hair,Lung" Chronic obstructive pulmonary disease Prevent Histological staining//Cell morphological analysis//SA-β-gal activity assay//Flow cytometry "At 80 weeks of age, CD9/CD81 DKO mice were smaller and had less hair of a brownish color than WT mice, although DKO and WT mice could not be distinguished at 3 wk of age.Moreover, DKO mice developed progressive kyphosis and decreased bone mineral density. Muscle and visceral adipose tissue were significantly reduced in volume, as determined by CT quantitation.Consequently, DKO mice had remarkably shorter survival than WT mice. Histological examination revealed that DKO mice developed emphysema and osteoporosis at 80 wk. Moreover, Although the CD4/CD8 ratio, one of markers in immunosenesence, was not altered in younger mice, it was reduced in aged DKO mice in comparison with WT mice.Double knock-down (DKD) of CD9/CD81 in epithelial cells resulted in cells with a large flattened morphology, and the proportion of SA-β-Gal-positive cells increased." -- -- -- -- SIRT1 -- Western blot//ELISA//Knockdown "Notably, knockdown of CD9 and CD81 in epithelial cells additively downregulated the expression of SIRT1, whereas knockdown of CD151 did not. The reduced expression of SIRT1 was further verified by immunocytochemistry and ELISA ." Human HL delay aging 29572511 Sen_G_0549 CD81 975 protein coding A549 "Bone,Muscle,Adipose,Hair,Lung" Chronic obstructive pulmonary disease Prevent Histological staining//Cell morphological analysis//SA-β-gal activity assay//Flow cytometry "At 80 weeks of age, CD9/CD81 DKO mice were smaller and had less hair of a brownish color than WT mice, although DKO and WT mice could not be distinguished at 3 wk of age .Moreover, DKO mice developed progressive kyphosis and decreased bone mineral density. Muscle and visceral adipose tissue were significantly reduced in volume, as determined by CT quantitation.Consequently, DKO mice had remarkably shorter survival than WT mice. Histological examination revealed that DKO mice developed emphysema and osteoporosis at 80 wk. Moreover, Although the CD4/CD8 ratio, one of markers in immunosenesence, was not altered in younger mice, it was reduced in aged DKO mice in comparison with WT mice.Double knock-down (DKD) of CD9/CD81 in epithelial cells resulted in cells with a large flattened morphology, and the proportion of SA-β-Gal-positive cells increased." -- -- -- -- SIRT1 -- Western blot//ELISA//Knockdown "Notably, knockdown of CD9 and CD81 in epithelial cells additively downregulated the expression of SIRT1, whereas knockdown of CD151 did not. The reduced expression of SIRT1 was further verified by immunocytochemistry and ELISA." Human HL delay aging 29572511 Sen_G_0550 FLT1 2321 protein coding HUVEC -- Alzheimer's disease Accelerate SA-β-Gal activity assay//Western blot "We show that overexpression of chimeric EGLT-VEGFR-1 in HUVECs for 72 h resulted in robust induction of the senescent phenotype as measured by β-galactosidase staining, with or without stimulation with EGF. " -- -- -- -- p21-p53 Activation Western blot//qRT-PCR "Western blotting analysis from chimeric receptor EGLT-transfected lysates confirmed the increased expression of the VEGFR-1 protein and increased p21 proteins levels.qRT-PCR analysis showed increased mRNA levels of VEGFR-1, p21, and p53." Human L delay aging 30576228 Sen_G_0551 LMNA 4000 protein coding -- Mice Aging Prevent Knockdown//Immunostaining "The body weight of Lmna?/?mice began to decline at the 10th week of age, while the weight of wild mice continued to increase, showing that, at that stage, the Lmna?/? mice started to lose weight, indicating a decline in the metabolic state of their body.A signifcant increase in the number of p16INK4aexpressing cells was observed in the BAT of Lmna?/?mice (64.33±2.333% versus 50.33±2.603,P = 0.0161)." UCP1//beta3-AR//PRDM16 --// --// -- Western blot "UCP1 and beta3-AR protein levels in Lmna?/?mice were significantly lower than those in WT mice at 14 weeks of age (UCP1: 0.04740.0089versus 1.000±0.0666, P =0.0001; beta3-AR: 3143±0.0329 versus 1.000±0.0445, P = 0.0002).Both the protein and mRNA levels of PRDM16 were decreased at the age of 14 weeks in Lmna?/?mice." -- -- -- -- Human L delay aging 30116163 Sen_G_0552 DLX2 1746 protein coding BJ -- Aging Accelerate SA-β-gal activity assay We also confirmed the senescence bypass phenotype by staining for senescence-associated β-galactosidase (SA-β-Gal). -- -- -- -- p53-p21//ATM-p53 Downregulation//Downregulation Western blot "We examined the status of the phosphorylation mark of p53 activation on Ser15 and p21 expression level in both young and senescent cells. We found that DLX2 expression led to reduced p53 Ser15 phosphorylation and p21 expression. We found that 2 wk after Ras virus infection, hTERT immortalized BJ cells expressing DLX2 showed reduced activation of p53 and the DNA damage marker γH2AX . Consistent with our results with replicative senescence, DLX2 expression also led to reduced levels of ATM and DNA-PKcs protein ." Human HL delay aging 26833729 Sen_G_0553 SIRT1 23411 protein coding SH-SY5Y -- Alzheimer's disease Prevent MTT assay //CCK-8 assay We further found that siSIRT1 treatment resulted in inhibited cell proliferation as indicated by the MTT assay and the CCK8 assay in a time course monitoring days 1 to 7. p53//CREB --//-- Western blot We further found that siSIRT1 also inhibited CREB phosphorylation and enhanced p53 expression. PI3K-Akt -- Western blot//Immunofluorescence "Using the Western blotting assay, we observed that SIRT1 expression was absent in the siSIRT1-treated cells, but the PI3K levels were similar regardless of the treatment. AKT phosphorylation at the 308 site was inhibited by siSIRT1 treatment while the phosphorylation at the 473 site was similar among all of the three groups, indicating that SIRT1 regulates AKT phosphorylation, specifically at the 308 site.We next examined the subcellular localization of SIRT1 and AKT using immunostaining analysis, and the results showed that AKT was activated in these cells as indicated by plasma membrane localization." Human L delay aging 28962864 Sen_G_0554 SIRT1 23411 protein coding VSMC -- Abdominal aortic aneurysms Prevent SA-β-gal activity assay "As expected, the SA-β-gal activity assay also confirmed the antiaging effect of SIRT1 overexpression." p21 Downregulation SA-β-gal activity assay//Western blot//Knockdown "These results suggest that SIRT1 inhibited Ang II–induced p21 expression by deacetylation of p53 in AAAs. The increased p21 expression caused by Ang II was significantly greater in the aortas of aged mice than that in young mice. Similarly, increased p21 expression was found in human AAA samples.The results of SA-β-gal staining indicated that p21 knock-down not only blocked Ang II–induced VSMC senescence but also eradicated the promotional effect of SIRT1 knockout." -- -- -- -- Human HL delay aging 27650558 Sen_G_0555 CAV1 857 protein coding "WI-38,IMR-90" -- Aging Prevent SA-β-gal activity assay//Western blot//Knockdown "We found that down-regulation of caveolin-1 by shRNA was sufficient to induce cellular senescence in both WI-38 and IMR-90 cells, as quantified by senescence-associated β-galactosidase activity (SA-β-gal) staining and immunoblot analysis for p21, a marker of cellular senescence." IFT88//AURKA --//-- Western blot//Immunostaining//SA-β-gal activity assay "More specifically, because down-regulation of IFT88 is known to inhibit ciliogenesis, IFT88 protein expression was downregulated by siRNA in caveoli n-1–lacking WI-38 cells. We found that, when primary cilia formation was prevented by IFT88 down-regulation in caveolin-1–lacking cells, cellular senescence was dramatically inhibited .To this end, caveolin-1 deficiency was achieved by shRNA in both WI-38 and IMR-90 cells, and the protein levels of AURKA were quantified by immunoblotting analysis. AURKA was virtually lost in caveolin-1 lacking cells." -- -- -- -- Human L delay aging 30596512 Sen_G_0556 NBR1 4077 protein coding MCF-7 -- Aging Prevent Cell morphological analysis//SA-β-gal activity assay//BrdU assay//Western blot "In MCF-7 cells, cellular senescence was induced after NBR1 siRNA transfection, as determined by cellular morphologies, cell counts, SA-β-gal staining, BrdU incorp- oration,and expression levels of p53 and p21WAF1/CIP1(p21) proteins and IL-6 and -8 mRNAs." p53//p21//p38 --//--//Downregulation Western blot//SA-β-gal activity assay//Knockdown "Cellular senescence induced by NBR1 abrogation p53- and p21-dependent. Inhibition of p53 by RNAi or pifithrin-a treatment prevented senescence. In addition, NBR1 abrogation induced senescence in p53 wild-type (WT) HCT-116 cells, but not in isogenic p53-null HCT-116 or p53-null PC3 cells. Knockdown of p21 also prevented cellular senescence .NBR1 down-regulated p38 activity in that basal and anisomycin-induced activation of p38 was enhanced by NBR1 abrogation, but was reduced by overexpression of NBR1." ERstress-ATF6a -- Knockdown//Western blot//SA-β-gal activity assay "Knockdown of ATF6a suppressed cellular senescence, as determined by cellular morphologies, cell counts, SA-β-gal staining, and expression levels of p53 and p21 proteins .In addition, transcription by ATF6a increased after NBR1 abrogation as demonstrated by a reporter assay. BiP expression, ATF6a cleavage, eIF2a phosphorylation and X-box binding protein (XBP)1 splicing were all upregulated .Moreover, treatment with the ER stress inhibitor, salubrinal,attenuated cellular senescence. Oxidative stress triggered NBR1 abrogation-induced ER stress because the ER stress was prevented by an antioxidant (Tiron) or inhibitors of NOX (DPI and AEBSF), or by knockdown of NOX2 and -4." Human L delay aging 30260700 Sen_G_0557 SIRT1 23411 protein coding VSMC -- Diabetes Prevent SA-β-gal activity assay//Western blot//qRT-PCR "Senescence was evident at day 4 with an inverse correlation (p < 0.0359) (r2 = 0.2469) between SIRT1 levels and the presence of senescent cells.Furthermore, there was a significant increase in the level of cellular senescence under osteogenic conditions compared to untreated control cells (p < 0.0417), and a further increase in the numbers of SA-βgal positive cells when exposed to hyperglycaemic osteogenic conditions, compared to the osteogenic conditions (p < 0.0094).Upstream of p21, p53 mRNA was also increased in all treatments where SIRT1 is inhibited (p < 0.0232)." -- -- -- -- RUNX2 Downregulation ChIP//Western blot//qRT-PCR "The acetylation profile of the RUNX2 promotor was measured via ChIP qPCR. RUNX2 promotor acetylation was significantly reduced in the hyperglycaemic conditions with the addition of SRT1720, suggesting a decrease in RUNX2 transcription was a direct result of SIRT1 activation . Furthermore, SRT1720 activation of SIRT1 activity resulted in a reduction in RUNX2 mRNA under osteogenic conditions, with a further significant reduction in RUNX2 expression under hyperglycaemic conditions (p < 0.0231), compared to the untreated cells. The decrease in RUNX2 mRNA expression after SIRT1 activation correlates with a decrease in RUNX2 protein expression in both osteogenic and hyperglycaemic conditions at day 4. Conversely, RUNX2 protein was increased following inhibition of SIRT1." Human L delay aging 30696833 Sen_G_0558 SCN9A 6335 protein coding HEC -- Aging Accelerate SA-β-gal activity assay//RT-qPCR//Knockdown "As expected, inducing the oncogenic stress (+4-OHT) resulted in proliferation arrest, as demonstrated by their reduced ability to form colonies and by the decrease in the level of the proliferation marker KI67,while it led to an increase in the SA-β-Gal activity and in the expression of two SASP components IL8 and IL6,both major hall-marks of senescence. Strikingly, the knockdown of SCN9A in HEC-TM cells overcame all of the hallmarks of senescence induced by an oncogenic stress." NF-kB -- qRT-PCR//Immunofluorescence "We examined whether or not the inhibition of NF-KB transcription factors, either by constitutively expressing a stabilized version of IKBA (mIKBA), a well-known inhibitor of NF-KB, or by knocking down the expression of RELA, the main subunit of the NF-KB transcription factors, blocked the induction of SCN9A during OIS. Both approaches significantly reduced the induction of SCN9A following an oncogenic stress at the mRNA, as well as at the protein levels." Ca-Rb-E2F -- qRT-PCR//Live calcium imaging The inhibition of Rb by E7 prevented the repression of mitotic genes induced by plasma membrane depolarization and blocked plasma membrane depolarization-induced senescence.We observed increased calcium after plasma membrane depolarization in HEC-T cells. Human HL delay aging 29446526 Sen_G_0559 CST1 1469 protein coding "MDA-MB-231,SW480 " -- Aging Prevent Knockdown//SA-β-gal activity assay//RT- PCR//Western blot "Following CST1 knockdown, cell populations exhibiting SA-β-gal-positive staining increased to 70–80% and 55–90% in MDA-MB-231 and SW480 cells, respectively. Notably, the gene expression of representative SASP genes, including interleukin-6 (IL-6) and chemokine C-C motif ligand 20 (CCL20), was induced by CST1 knockdown in MDA-MB-231 and SW480 cells.To confirm that the G0/G1-phase cell cycle arrest was caused by CST1 knockdown, we conducted western blotting and found that CST1 knockdown suppressed cyclin D1 and phospho-retinoblastoma (p-Rb) and induced p21." CatB -- Knockdown//SA-β-gal activity assay//Western blot "CST3 knockdown rescued extracellular CatB activity and significantly inhibited SA-β-gal activity in CST1 knockdown MDA-MB-231 cells.We found that CST1 knockdown suppressed extracellular, but not intracellular, CatB activity." GSK3β -- Knockdown//SA-β-gal activity assay//Western blot "The increased GSK3β phosphorylation caused by CST1 knockdown was inhibited by the addition of rCys-SN. Although SA-β-gal activity induced by CST1 knockdown was unaltered in mock vector and wild-type GSK3β-expressing cells, the ectopic expression of GSK3β-S9A (Activate form) significantly suppressed the SA-β-gal activity induced by CST1 knockdown . CatB knockdown also induced the inhibitory phosphorylation of GSK3β at serine 9." Human L delay aging 28383558 Sen_G_0560 ATF6 22926 protein coding NHDF -- Aging Accelerate Knockdown//SA-β-gal activity assay Only ATF6α knockdown significantly reduced the number of SA-β-Gal positive-cells upon DTT treatment. -- -- -- -- COX-2-PGE2 Activation ELISA//qRT-PCR//Knockdown//Western blot "ELISA assays showed that the increase in PGE2 synthesis and secretion induced by DTT was totally abolished upon ATF6 knocked-down cells .Knockdown of ATF6 and IRE1 but not PERK, reduced COX2 mRNA and protein levels at senescence, as well as the production and secretion of PGE2." Human L delay aging 28803844 Sen_G_0561 SIRT1 23411 protein coding VSMC Plaques and normal vessel Atherosclerosis Prevent qPCR//Western blot "SIRT1 mRNA was significantly decreased in the media of plaques versus normal vessels, associated with significantly increased p16ink4.SIRT1 mRNA and protein expression were reduced in plaque VSMCs and senescent aortic VSMCs versus early-passage normal human VSMCs." -- -- -- -- -- -- -- -- Human L delay aging 23224247 Sen_G_0562 CEBPG 1054 protein coding MEF -- Aging Prevent Cell morphological analysis//SA-β-gal activity assay//qRT-PCR//Knockdown "MEF cultures contained many cells with a flattened morphology and vacuolated cytoplasm, indicative of premature entry into senescence. This observation was confirmed by SA–β-Gal staining assays, which showed that mutant MEFs contain ～4-fold more senescent cells than WT MEFs.We used qPCR to evaluate expression of candidate SASP genes (GROα/Cxcl-1, Cxcl2, Ccr-1, Il6, Il1a, and Il1b)in Cebpg-/-MEFs and RasV12-expressing Cebpb-/-MEFs. Each gene was induced in C/EBPγ-deficient MEFs compared to WT cells." C/EBPβ -- Knockdown//SA-β-gal activity assay//qRT-PCR "C/EBPβ knockdown with two independent shRNAs increased the proliferative capacity of Cebpg-/-MEFs, as judged by cell densities 7 days after plating. C/EBPβ ablation also significantly reduced the proportion of senescent cells and reversed the aberrant expression of SASP genes Cxcl1 and Cxcl2." -- -- -- -- Human HL delay aging 23775115 Sen_G_0563 SIRT1 23411 protein coding HUC-F2 -- Aging Prevent SA-β-gal activity assay Results clearly showed that SIRT1 significantly promoted proliferation and prevented replicative senescence HUC-F2 cells. c-Myc//hTERT --//-- qRT-PCR//CHIP//Luciferase reporter assay "The results indicated that SIRT1, as well as starvation conditions, increased the promoter activity of c-MYC [11] and the transcription of c-MYC gene. Further, we observed the increased amount of c-MYC recruited to the hTERT promoter. The results showed that SIRT1 significantly increased the transcriptional activation ability of c-MYC.Results showed that SIRT1, but not SIRT1-HY, increased hTERT transcription, as evidenced by the promoter assay and quantitativeRT-PCR (qRT-PCR)." -- -- -- -- Human L delay aging 22197555 Sen_G_0564 FOXQ1 94234 protein coding 2BS -- Esophageal cancer Prevent DAPI staining//SA-β-gal activity assay//Flow cytometry "Meanwhile, miR30-FOXQ1 also resulted in emerging the morphological features of senescence, characterized by enlarged and flattened cell size, increased senescenceassociated heterochromatin foci, elevated activity of senescence-associated β-galactosidase (SA-β-gal), a biomarker for senescent cells, and reduced S and increased G1 compartments compared with scramble control vector.Conversely, LPC-FOXQ1 induced much lower SA-β-gal activity and less cell cycle progression than the infection with its corresponding empty control vector." SIRT1//IL-6//IL-8//p16 Binding//Downregulation//Downregulation//Downregulation Western blot//qRT-PCR//CHIP-qPCR "Western blot analysis revealed that FOXQ1 overexpression significantly increased the protein level of SIRT1 in HEK293T cells.In parallel with the findings from western blot analysis, qRT-PCR analysis also showed a positive regulation of SIRT1 mRNA expression by FOXQ1,indicating that FOXQ1 could activate transcription of SIRT1.The ChIP-qPCR results indicated a significant enrichment of FOXQ1 in the promoter of SIRT1 in the FOXQ1 overexpressed cells compared with the control cells. LPC-FOXQ1 markedly decreased the mRNA abundance of IL-6 and IL-8 compared with control transfection . Besides, increased FOXQ1 expression resulted in a decreased level of p16INK4aprotein, while removal of FOXQ1 exhibited an opposite effect on p16INK4aprotein level." NF-κB Upregulation Western blot "We found that the protein level of IκBα, an inhibitor of NF- κB, positively correlated with FOXQ1 and SIRT1.Western blot results revealed that the FOXQ1-induced upregulation of IκBα level was abolished by addition of EX-527 treatment.These results suggested that the FOXQ1-mediated regulation of SASP factors was dependent on SIRT1-NF-κB pathway." Human L delay aging 28726780 Sen_G_0565 SIRT1 23411 protein coding HUVEC -- Diabetic vascular complication Prevent SA-β-gal activity assay//Knockdown "Moreover, Knockdown of SIRT1 not only suppressed the deacetylase activity of SIRT1,but also increased the percentage of SA β-Gal staining in both RSV- and MET-treated cells." -- -- -- -- -- -- -- -- Human L delay aging 26629991 Sen_G_0566 PRKAA1 5562 protein coding HUVEC -- Diabetic vascular complication Prevent SA-β-gal activity assay//Western blot "It was shown that silencing of AMPK expression diminished SIRT1 activation and its downstream signaling.Moreover, it abolished the protective effects of RSV and MET against enhanced oxidative stress and accelerated cellular aging." SIRT1 -- Western blot It was shown that silencing of AMPK expression diminished SIRT1 activation and its downstream signaling. -- -- -- -- Human L delay aging 26629991 Sen_G_0567 NRAS 4893 protein coding Melanocyte -- Aging Accelerate Immunostaining//Cell morphological analysis//SA-β-gal activity assay//DAPI staining//SAHF "As expected, 15 days post-transduction the majority of N-RASQ61K transduced melanocytes displayed several markers of oncogene-driven senescence, namely cell flattening, increase in cellular size, significantly reduced Ki67 expression, increased SA-β-Gal activity and the formation of SAHF." -- -- -- -- MAPK//AKT//p16INK4a-pRb//p53-p21Waf1 Activation//Activation//Activation//Activation Western blot "N-RASQ61K induced melanocyte senescence was also associated with activation of the MAPK and AKT pathways, as shown by the increased phosphorylation of ERK (p-ERK), and AKT (p-AKT) at 5,10 and 15 days post infection.In addition, expression of oncogenic N-RAS led to p53 induction, increased expression of the p16INK4a and p21Waf1 cyclin dependent kinase inhibitors and reduced accumulation of pRb phosphorylated at serine residues -807 and -811 (p-pRb).These data suggest that the activation of pRb is the dominant effector of oncogene-induced melanocyte senescence." Human L delay aging 20157537 Sen_G_0568 TGFB1 7040 protein coding U937 -- Cancer Accelerate SA-β-gal activity assay//qRT-PCR//SAHF "While SAHF and increased p16 expression was observed already after 3 days, SA-β-Gal was not apparent until 6 days of treatment. No signs of increased proliferation was observed during continued culture during a couple of weeks." Mad1//Myc Upregulation//Downregulation Western blot "To investigate the effects of TGF-β1 on Myc/Max/Mad network protein expression, the respective proteins were immunoprecipitated from 35S-labeled U-937-myc-6 cell extracts . The synthesis of Mad1 increased substantially;meanwhile, the synthesis of c-Myc decreased after TGF-β1 treatment. A slight decrease in v-Myc synthesis was also observed while no major change in the expression of Max occurred." -- -- -- -- Human L delay aging 19766114 Sen_G_0569 BAZ1A 11177 protein coding "A549,U2OS" -- Cancer Prevent CCK-8 assay//Flow cytometry//SA-β-gal activity assay//Knockdown//EdU assay "CCK-8 assay showed BAZ1A-KD cells had decreased proliferation rate compared to control cells.In addition,cell cycle was arrested at G1 phase in BAZ1A-KD cells. Noteworthy, all of the five BAZ1A-KD cell lines showed increased percentage of positive SA-β-Gal stained cells. Moreover, reduced EdU incorporation rate was also observed in BAZ1A-KD cells , reflecting decreased level of DNA synthesis, which is also a well-known molecular phenotype of senescent cells." SMARCA5 -- Western blot//CCK-8 assay//Flow cytometry//SA-β-gal activity assay//Knockdown "To examine whether SMARCA5 can regulate the abundance of BAZ1A and then influence senescence, we stably knocked down SMARCA5 by lentivirus mediated short hairpin RNA in A549 and U2OS cells, and reduced BAZ1A protein levels were found in SMARCA5-KD cells, which also exhibited senescenceassociated phenotypes, including decreased proliferation rate, cell cycle arrest at G1 phase, and increased percentage of positive SA-β-Gal stained cells." Smad3-p21 -- ChIP-qPCR//qRT-PCR//Western blot "The results showed enriched signal of BAZ1A binding to the promoter region of SMAD3 compared to non-specific IgG binding control, which was further validated by Chromatin Immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) .Our data showed the upregulation of both SMAD3 and CDKN1A in BAZ1A-KD cells,indicating upregulated SMAD3 activated the expression of CDKN1A and led to slower cell proliferation and ultimately cellular senescence." Human HL delay aging 31085244 Sen_G_0570 RRM2B 50484 protein coding IMR-90 -- Aging Prevent SA-β-gal activity assay//Western blot "Unexpectedly, the silencing of RRM2B triggered premature senescence in young IMR90 cells. The specific silencing of RRM2B led to the progressive accumulation of multiple senescent regulators, including p53, p21CIP1 and p16INK4A, from day 7 to 19." -- -- -- -- p38 MAPK//p53 --//-- SA-β-gal activity assay//Western blot//BrdU assay "p38MAPK phosphorylation was profoundly increased when RRM2B was silenced compared to shRRM2Bmut-expressing cells. Downstream targets of p38MAPK were also activated, as indicated by the significant elevation of phosphorylated MAPKAPK-2, a substrate of p38MAPK, and the phosphorylation of HSP27, a substrate of MAPKAPK-236.Interestingly, the silencing of p53 by shRNA in shRRM2Bexpressing cells was sufficient to rescue premature senescence, as indicated by the significant reduction in SA-β-gal activity and the increase in the replication index .?Cells expressing both shRRM2B and shTP53 showed reduced expression of p21CIP1 and increased levels of RRM1 and RRM2 , whereas p16INK4A was unchanged compared to cells expressing shRRM2B alone." Human HL delay aging 23139867 Sen_G_0571 NR2E1 7101 protein coding "LNCaP,DU 145" -- Prostate cancer Prevent SA-β-gal activity assay SA-β-Gal analysis revealed that there was a significant reduction of SA-β-Gal-positive cells in LNCaPTLX infectants compared to LNCaP-pBABE infectants.Cytochemical analysis on SA-β-Gal activity revealed that there was a dramatic increase of SA-β-Gal-positive cells in both LNCaP-shTLX and DU145-shTLX infectants as compared to Scramble-shRNA infectants. p21//SIRT1 Downregulation//Upregulation Western blot//CHIP "Immunoblot analysis of senescence-associated markers showed that the expression level of cyclin-dependent kinase inhibitor p21WAF1/CIP1 (hereafter as p21) was markedly suppressed or undetected in LNCaP-/PC-3-TLX infectants but became upregulated in shTLX-infectants of both LNCaP and DU145 cells.ChIP analysis performed in prostate cancer cells identified that there was a significant enrichment of TLX at one consensus TLX-binding motif (TTCAGT) located at - 656 -650 bp and also a palindromic sequence (ACTGAA) at -616 -610 bp in the proximal CDKN1A (p21) promoter.Interestingly, we also detected a significant elevation of protein expression of an NAD- dependent protein deacetylase Sirtuin-1 (SIRT1) in LNCaP-TLX infectants but a reduction in shTLX-infectants by immunoblot analysis.ChIP assay showed that a proximal region of human SIRT1 gene promoter (-129 ? +32 bp) could be immunoprecipitated in TLX- and vector-transfected prostate cancer cells." -- -- -- -- Human L delay aging 25557355 Sen_G_0572 ALPL 249 protein coding MSC Adipose Aging Prevent SA-β-gal activity assay//Western blot "Specifically, the number of SA-β-gal+ cells and the expression of p16 were obviously increased in the Alpl+/- BM, but TERT expression was decreased relative to that in the WT controls." ATP -- RadioActivate ATP assay "After the induction, the Alpl+/- MSCs, but not the WT MSCs, released a large amount of ATP. More strikingly, the downregulation of Alpl led to increased extracellular ATP in the WT MSCs, whereas enforcing expression in the Alpl+/- MSCs significantly reduced the extracellular ATP level, suggesting that Alpl in MSCs probably regulates ATP release." AMPKα -- Western blot "The supernatant of the Alpl+/- MSCs suppressed the expression levels of p-AMPKα and p-ACC in 293T cell lines, indicating that the higher extracellular ATP levels due to the Alpl deficiency could inactivate the AMPKα pathway." Human HL delay aging 30210899 Sen_G_0573 CAV1 857 protein coding A549 -- Aging Prevent Cell morphological analysis//SA-β-gal activity assay//Knockdown//Cell counting//Colony formation assay "Unexpectedly, Cav-1 knockdown increased β-gal positivity and changed the cellular morphology from a normal epithelial shape to a fried egg-like shape.Cav-1 knockdown significantly decreased cellular proliferation and colony-forming capacity compared with the si-control-treated cells." SIRT1 -- Western blot//Knockdown "Therefore, we measured SIRT1 activity by measuring acetylated p53 expression after Cav-1 knockdown in A549 cells using immunoblotting. The acetylation of p53 was gradually increased with time after Cav-1 knockdown." p53-p21 -- SA-β-gal activity assay//Knockdown//Western blot//Cell counting "Analyses of cell numbers, β-gal staining positivity, and p-pRb, p53, and p21 expression levels showed that p53 or p21 knockdown prevented Cav-1 knockdown-induced cellular senescence." Human L delay aging 28514055 Sen_G_0574 TBX3 6926 protein coding MEF -- Ulnar-mammary syndrome Prevent Histological staining "TBX3 is able to immortalize MEF cells, suggesting inhibition of senescence.Cells infected with pFB-Neo-TBX3 have passed 15 passages without senescence." -- -- -- -- -- -- -- -- Human HL delay aging 15289316 Sen_G_0575 TBX3 6926 protein coding MEF -- Ulnar-mammary syndrome Accelerate Histological staining "In contrast, in cells harboring TBX3+2a, acceleration of senescence occurs, and growth is slower than in the control (pFB-Neo). Cells infected with pFBNeo and pFB-Neo-TBX3+2a stopped growing at eight and six passages, respectively." -- -- -- -- -- -- -- -- Human HL delay aging 15289316 Sen_G_0576 TAT 6898 protein coding MSC -- AIDS Accelerate SA-β-gal activity assay//Western blot "In control cells, the percentage of senescent cells X-gal stained was 8.6±2.2% and 9.3 ± 2.2%, after 10 and 20 days of culture, respectively. After 10 days, senescence was increased in cells treated with Tat+Nef, and after 20 days, its level was of 17.1 ± 1.6%, 18.0 ± 4.1% and 20.2 ± 3.7% in Tat-, Nef- and Tat+Nef-treated cells, respectively." -- -- -- -- NF-κB Activation Western blot "Treatment with Tat but not Nef resulted in an increased nuclear translocation of the activated pro-inflammatory and pro-senescent transcription factor NF-κB, as shown by the accumulation of the activated phospho-Ser 536 form of p-65 in the nucleus ." Human L delay aging 25847297 Sen_G_0577 nef 156110 protein coding MSC -- AIDS Accelerate SA-β-gal activity assay//Western blot "In control cells, the percentage of senescent cells X-gal stained was 8.6±2.2% and 9.3 ± 2.2%, after 10 and 20 days of culture, respectively. After 10 days, senescence was increased in cells treated with Tat+Nef, and after 20 days, its level was of 17.1 ± 1.6%, 18.0 ± 4.1% and 20.2 ± 3.7% in Tat-, Nef- and Tat+Nef-treated cells, respectively." Beclin1 Downregulation Immunostaining//Co-IP "The cellular oxidase activity, was unchanged after 10 days of treatment with the HIV proteins, but increased after 20 days by 1.3- to 1.4-fold by Tat and/or Nef, when compared to control cells along with an increased superoxide dismutase activity (SOD).Moreover, we observed a direct interaction between Nefand Beclin-1, which were co-immunoprecipitated, suggesting that Nef could inhibit autophagy through a direct interaction with Beclin-1." -- -- -- -- Human L delay aging 25847297 Sen_G_0578 GDF15 9518 protein coding Aortic endothelial cell -- Atherosclerosis Accelerate SA-β-gal activity assay Upregulation of GDF15 caused a decrease in cell proliferation and an increase in SA-β-gal staining compared with the control lentivirus-transduced cells. ERK Activation Western blot//Immunostaining "We noted that ERK phosphorylation was increased following the rhGDF15 protein treatment. In addition, IR-induced ERK activation was controlled by the downregulation of GDF15.ROS generation was increased in GDF15-tranduced cells compared to control virus-transduced cells." p16-Rb Upregulation SA-β-gal activity assay//Cell counting//Knockdown//Western blot "On the contrary, the overexpression of GDF15 had no significant effects on cell proliferation in the p16 knockdown cells . The measurement of SA-β-gal activity indicated that p16 knockdown inhibited GDF15-induced cellular senescence, but p53 knockdown did not.Increased expression of GDF15 induced p16 expression and treatment with GDF15 recombinant protein increased p16 mRNA by approximately 2.5 fold. Both endogenous and exogenous GDF15 protein increased p16 protein and decreased the phosphorylation of Rb, which causes its detachment from E2F transcription factor." Human HL delay aging 26909594 Sen_G_0579 PAK4 10298 protein coding Hs 578T -- Breast cancer Prevent Cell morphological analysis//SA-β-gal activity assay//Western blot//BrdU assay "After transient transfection of two independent small interfering RNAs (siRNAs) targeting the human PAK4 gene, Hs 578T breast cancer cells adopted a flatter and larger senescenceassociated morphology and exhibited elevated SA-β-gal activity (as measured with the two substrates X-Gal29and MUG30) that was accompanied by a significant decrease in BrdU-incorporation.Genes involved in cell cycle arrest, DNA damage/ repair, and SASP factors are typically upregulated in senescent cells. PAK4 knockdown also increased protein expression of the known senescence-regulators p53 and p21." RELB Downregulation Western blot "This inverse correlation was also observed at the protein level in Hs 578T breast cancer cells where PAK4 knockdown upregulated RELB. Considering expression as continuous vari- ables, the expression of PAK4 and RELB displayed the strongest significant inverse association ." NF-κB Downregulation qRT-PCR "PAK4 inhibits NF-κB signaling.Upregulation of several NF-κB target genes upon PAK4 knockdown was validated by RT-qPCR in Hs 578T cells, including the NF-κB subunits NFKB1, NFKB2, and RELB as well as the previously characterized NF-κB response genes CD82, S100A4, TIMP2, CDKN1C, PRKCD, TWIST1, SPP1, TP53, and TRAF2." Human HL delay aging 31399573 Sen_G_0580 CCND1 595 protein coding MCF-7 -- Breast cancer Prevent Cell morphological analysis//SA-β-gal activity assay//Immunostaining//SAHF//Knockdown "Cyclin D1-depleted cells appeared flattened with large volumes of cytoplasm and were positive for SA-β-Gal staining. Cyclin D1-depleted cells also showed a significant increase in senescence-associated heterochromatin foci (SAHF), another putative marker for cellular senescence (Narita et al., 2003)." -- -- -- -- p38-FOXO3a-p27 -- Western blot//Immunostaining//Cell counting "We detected upregulation of phospho-p38 (Thr180/Tyr182) and JNK-mediated c-JUN phosphorylation at Ser73 in cyclin D1- depleted cells around 48-72 h after cyclin D1 depletion, but not in control cells.We detected an upregulation of FOXO3a protein in associatio nwith increased FOXO3a Ser7 phosphorylation, which is involved in the stress-induced activation of FOXO3a by p38 and translocalization of FOXO3a from the cytoplasm to the nucleus after cyclin D1 depletion. We found that p27 protein level was also upregulated in cyclin D1-depleted cells, whereas GADD45a and SOD2 were not upregulated at the protein level ." Human L delay aging 29880532 Sen_G_0581 FBXO46 23403 protein coding MCF-7 -- Aging Prevent SA-β-gal activity assay//Knockdown "It was observed that depletion of FBXO46 significantly increased the population of senescent cells, which was inhibited upon co-depletion with FBXO31." FBXO31 Downregulation Western blot//Co-IP "Immunoblot analysis revealed the presence of FBXO46 in the FBXO31 immunoprecipitates. In a reciprocal co-immunoprecipitation assay, FBXO31 was found to be present in the FBXO46 immunoprecipitates, suggesting that FBXO46 and FBXO31 interact with each other.The results showed that FBXO46 significantly decreased FBXO31 levels in a dose- dependent manner." -- -- -- -- Human L delay aging 30171069 Sen_G_0582 POT1 25913 protein coding "MRC-5,WI-38,NHF" -- Aging Prevent SA-β-gal activity assay//Knockdown//BrdU assay "The shRNA knockdown of endogenous POT1v1 or POT1v5 induced cellular senescence, characterized by cell growth arrest and SA-β-Gal activity, in normal human fibroblast strains MRC-5, NHF, and WI-38.The significant decrease in bromodeoxyuridine incorporation was associated with sh-v1 induced shsenescence(1.9% ,compared with 43.9% in control cells）." -- -- -- -- p53//p16 --//-- Western blot "The p53 dependence of sh-v5–induced senescence in this experiment was consistent with the Western blot results of cellular senescenceregulatory factors in normal human fibroblasts. Both sh-v1 and sh-v5 led to the increase in Ser15-phosphorylated p53 and the up-regulation of p21WAF1, an effector of p53-mediated cellular senescence.In contrast, only sh-v1 induced the expression of p16INK4A, another major effector for cellular senescence in human cells." Human L delay aging 18089797 Sen_G_0583 POT1 25913 protein coding "MRC-5,WI-38,NHF" -- Aging Prevent SA-β-gal activity assay//Knockdown//BrdU assay "The shRNA knockdown of endogenous v1 or v5 induced cellular senescence, characterized by cell growth arrest and SA-β-Gal activity, in normal human fibroblast strains MRC-5, NHF, and WI-38.The significant decrease in bromodeoxyuridine incorporation was associated with sh-v5–induced senescence (2.0%,compared with 43.9% in control cells）." -- -- -- -- p53 -- Western blot "The p53 dependence of sh-v5–induced senescence in this experiment was consistent with the Western blot results of cellular senescenceregulatory factors in normal human fibroblasts. Both sh-v1 and sh-v5 led to the increase in Ser15-phosphorylated p53 and the up-regulation of p21WAF1, an effector of p53-mediated cellular senescence." Human L delay aging 18089797 Sen_G_0584 MYC 4609 protein coding IMR-90 -- Aging Accelerate Crystal violet assay//SA-β-gal activity assay//BrdU assay//qRT-PCR//GSEA analysis "Expression of the reprogramming factors (OSKM) in IMR90 human fibroblasts causes a senescence-like growth arrest that constitutes an intrinsic barrier to reprogramming (Banito et al. 2009). Similar to oncogenic RASG12V, the expression of OSKM induces the cyclin-dependent kinase (CDK) inhibitors (CDKIs) p15INK4b, p16INK4a, and p21CIP1, which are involved in implementing the stable growth arrest associated with senescence. Gene set enrichment analysis (GSEA) found signatures for senescence and the SASP significantly enriched in the transcriptome of cells expressing OSKM." CDKN1A//MYOT//mTOR//UBE2E1 --//--//--//-- Crystal violet staining//SA-β-gal activity assay//BrdU assay "The ability of shRNAs targeting CDKN1A, MYOT, MTOR, and UBE2E1 to prevent OSKM-induced senescence was confirmed by increased proliferation, a higher percentage of cells incorporating BrdU, and a decrease in the percentage of senescence-associated βgalactosidase (SA-β-Gal)-positive cells when compared with IMR90 cells infected with OSKM and a control vector ." TGFβ -- BrdU assay//Follow-up analysis "Moreover, inhibition of TGFBRI signaling blunted the growth arrest triggered by OSKM. In this regard, the scRNA-seq data and the follow-up analysis highlighted that TGF-β signaling was induced by OSKM ." Human HL delay aging 29138277 Sen_G_0585 BRD4 23476 protein coding MKN28 -- Gastric cancer Prevent SA-β-gal activity assay//Knockdown//Western blot "Depletion of Brd4, but not Brd2 and Brd3, increased the number of SA-β-Gal-positive cells.In line with increased SA-β-Gal activity, the levels of p21 were enhanced in Brd4 knockdown cells." -- -- -- -- E2F-miR-106b-p21 -- RT-PCR//Luciferase reporter assay//CHIP//Western blot "Depletion of Brd4 or treatment of MKN28 cells with JQ1 up-regulated p21 mRNA levels with 2 3 folds induction in Brd4 knockdown cells and less than 2 folds induction in JQ1-treated cells.Consistently, depletion of Brd4 also increased the activity of 3 -UTR of p21 reporter. In contrast, overexpression of BRD4 in MKN28 cells decreased the luciferase activity of p21 3 -UTR luci- ferase reporter. When miR- 106b-5p and miR-519d-3p mimics were transfected into MKN28 cells followed by JQ1 treatment, miR-106b-5p but not miR-519d-3p mimics reduced the JQ1-induced cellular levels of p21, indicating that miR-106b targets p21 mRNA in MKN28 cells. Treatment of MKN28 cells with HLM006474 efficiently inhibited the binding of BRD4 to the promoter of miR-106b-5p,suggesting that E2F regulates the recruitment of BRD4 to the promoter of miR-106b-5p. Importantly, HLM006474 also down-regulated the expression of MCM7 and the expression of miR-106b-5p." Human L delay aging 29434197 Sen_G_0586 EZH2 2146 protein coding SGC-7901 -- Gastric cancer Prevent SA-β-gal activity assay//Flow cytometry//Knockdown EZH2 depletion cells expressed SA-β-gal and flow cytometric analysis demonstrated that the proportion of cells in the G2/M phase increased.We observed a significant inhibition in cellular proliferation in cells infected with lentivirus or EGCG compared with control treated. p21//p16 --//-- qRT-PCR//CHIP "Depletion of EZH2 elevated the expression of p21 and p16 in SGC-7901 cells. The effect of loss of EZH2 upon induction of p21 and p16 expression was transcriptional, since the transcript of CDKN1A and CDKN2A, which encodes p21 and p16, respectively, was upregulated in SGC-7901 that exhibited increases in p21 and p16 protein levels." -- -- -- -- Human L delay aging 24588771 Sen_G_0587 TNF 7124 protein coding ISO-HAS -- Aging Accelerate SA-β-gal activity assay//RT-PCR "Treatment with 10 ng/mL TNF-α for 48 h increased the proportion of SA-β-gal-positive cells in the endothelial cell culture.Gene expression of SHC1 and GLB1 increased at TNF-α concentrations above 1 ng/mL,and the significance of this effect was reached for all three genes at 10 ng/mL." p21 Upregulation Western blot We used Western blotting to examine the protein expression levels of p21 in endothelial cells following 10 ng/mL TNF-α treatment for 8 h. TNF-α exposure induced the expression of p21 in ISO-HAS cells. -- -- -- -- Human L delay aging 26802937 Sen_G_0588 PTEN 5728 protein coding MEF -- Prostate cancer Prevent SA-β-gal activity assay//Western blot "Pretreatment of Ptenlx/lx MEFs with aphidicolin,followed by acute inactivation of Pten,resulted in increased β-gal senescence staining and p53 accumulation after 24 hours." -- -- -- -- PI3K-mTOR -- SA-β-gal activity assay//Western blot "Surprisingly,while?this?procedure?resulted?in?efficient?Pten?loss,?senescence?and?p53?levels?were?completely?abolished?by?rapamycin.?Wealso?tested?genetically?whether?mTOR?is?also?essential?for?PICS,? taking?advantage?of?Pten–/–mTOR–/–?double-null?MEFs,and?found this to?be?true." Human L delay aging 20197621 Sen_G_0589 E2F7 144455 protein coding IMR-90 -- Aging Prevent BrdU assay//Colony formation assay//Cell counting "Still, in marked contrast, cosuppression of E2F7 together with RB was sufficient to bypass senescence, as measured by various proliferation assays such as BrdU incorporation, colony formation, and cell counting." -- -- -- -- p53 Binding CHIP-Seq "Interestingly, E2F7 has a p53-binding site in its promoter, and indeed, analysis of chromatin immunoprecipitation (ChIP)/next-generation sequencing (ChIP-seq) data from IMR90 cells under different growth conditions revealed a marked and specific binding of p53 to this site in senescence but not in growing conditions .p53 binds to the E2F7 promoter specifically during cellular senescence." Human HL delay aging 22802529 Sen_G_0590 TGFB2 7042 protein coding TM Eye Primary open-angle glaucoma Accelerate SA-β-gal activity assay//qRT-PCR "Exposure to TGF-β2 for 24 and 48 hours markedly increased the proportion of SA-β-Gal–positive TM cells to 31.6% ± 7.7% and 33.7% ± 5.4% of total cells.Treatment with TGF-β2 for 12, 24, and 48 hours increased three mRNA expressions of Apo J,SPARC,SM22." -- -- -- -- p16-Rb Activation qRT-PCR//Western blot "Treatment with TGF-β2 for 12 and 24 hours markedly increased the p16 mRNA expression by 1.7 ± 0.2- and 3.2 ± 0.2-fold, whereas exposure of cells to TGF-β2 for 48 hours did not influence p16 mRNA expression.Treatment with TGF-β2 for 24 hours markedly led to a downregulation of pRb to 42.1% ± 5.0% of the level of untreated control cells." Human L delay aging 20554622 Sen_G_0591 LMNA 4000 protein coding Fibroblast -- Lipodystrophic syndromes Accelerate SA-β-gal activity assay//Cell morphological analysis "In contrast, SA-β-galactosidase activity, assessed at pH 6, was absent in control cells up to passage 14, but present, even at early passages, in fibroblasts with LMNA mutations or treated with PIs." p16//p21 --//-- Western blot//Immunostaining "P16INK4aand p21WAF1,two cell cycle checkpoint inhibitors that participate in the setup of the senescence program,were overexpressed by 250–400% in fibroblasts bearing LMNA mutations at passages 9–15.Dichloro- fluorescein oxidation was increased two- to fivefold in fibroblasts with LMNA mutations at passages 6 8, as compared to control cells.Microscopic examination confirmed the increased production of ROS in fibroblasts with LMNA muta-tions,as compared to control fibroblasts." -- -- -- -- Human L delay aging 17612587 Sen_G_0592 NPM1 4869 protein coding "MIP101,RKO,HCT116" -- Colorectal cancer Prevent SA-β-gal activity assay//Knockdown "Based on SA-β-gal staining, there were more senescent cells among all CRC cell lines transfected with NPM1 siRNA compared to those transfected with scramble siRNA. By MUG assay, a 20%, 35% and 45% increase in cellular senescence were observed in MIP101, RKO and HCT116 cells following NPM1 siRNA knockdown, respectively." p53 -- Knockdown//Immunostaining//Western blot "Interestingly, remarkable increases in the expression of p53 were noted after NPM1 gene silencing in all three CRC cell lines by immunofluorescence staining and confirmed in MIP101 cells by immunoblotting." -- -- -- -- Human L delay aging 23536448 Sen_G_0593 ATRX 546 protein coding WD/DDLS -- Aging Accelerate SA-β-gal activity assay//SAHF//qPCR//Crystal violet assay "Reducing ATRX in these cells affect the accumulation of SA-β-gal-positive cells, SAHF-positive cells, the accumulation of three of the four mRNAs (CXCL1, GM-CSF, IL-6, and IL-8) that increase as part of the SASP in LS8817 cells, and the ability of the cells to return to the cell cycle following drug removal and replating." HRAS -- qRT-PCR//CHIP-seq "ATRX binding was strongly enriched at the HRAS locus in senescent LS8817 but not quiescent LS8107 cells.Consistent with the importance of ATRX for repression, HRAS expression increased when ATRX was knocked down in cells that were already senescent." -- -- -- -- Human HL delay aging 28855512 Sen_G_0594 SIAH1 6477 protein coding MRC-5 -- Aging Accelerate Knockdown "The Siah1 knockdown cells had increased proliferation rate in the early phase of the experiment and had an extended replicative lifespan, which was approximately 2 or 3 PDL longer than control cells." p53//TRF2 Upregulation//Downregulation Western blot//qRT-PCR "In these experiments, the expression of Siah1 protein was inversely correlated with the expression of TRF2 protein; Siah1 was decreased when TRF2 was increased,and Siah1 was increased when TRF2 was decreased.The overexpression of a stabilized form of Siah-1 resulted in the downregulation of TRF2.Decreased SIAH1 mRNA levels were also confirmed in cells with p53 knockdown.The overexpression of wild-type p53 led to increased Siah-1 and decreased TRF2." -- -- -- -- Human L delay aging 21057505 Sen_G_0595 MYC 4609 protein coding HFF -- Aging Accelerate SA-β-gal activity assay "Furthermore, while c-Myc overexpression facilitated cellular senescence, which was detected by βgalactosidase staining, this phenomenon was subdued following the depletion of USP10." p14//p53//USP10 --//--//Upregulation Western blot//qRT-PCR "Interestingly, p14ARF protein stability was increased by c-Myc overexpression. Accordingly, c-Myc also increased the protein stability of p53, a downstream target of p14ARF, through an indirect or a direct pathway.Corroborating these findings, cMyc overexpression increased USP10 protein levels as well as its mRNA in HFF and IMR90 cells." -- -- -- -- Human HL delay aging 29472714 Sen_G_0596 USP10 9100 protein coding "IMR-90,HFF" -- Aging Accelerate SA-β-gal activity assay//Western blot "USP10 overexpression stabilized p14ARF protein levels in both cell lines.In line with these observations, these cells exhibited accelerated cellular senescence with decreased cellular growth. These phenomena were prevented by p14ARF ablation under USP10 overexpression. Overall, these results indicated that USP10 could accelerate cellular senescence through p14ARF stabilization." p14 -- Western blot USP10 overexpression stabilized p14ARF protein levels in both cell lines. -- -- -- -- Human HL delay aging 29472714 Sen_G_0597 SUSD2 56241 protein coding Ishikawa -- Endometrial cancer Prevent SA-β-gal activity assay//Knockdown SiRNA-SUSD2 significantly increased blue staining indicative of SA-β-Gal activity and thus senescent cells. TGFβ//SMAD2/3//LGALS1 Downregulation//--//-- Western blot//Knockdown "At 72h TGFβ significantly decreased the number of SUSD2+ cells. The significant decrease of SUSD2 at protein level was paralleled by a similar decline of SUSD2 mRNA level.To our surprise, levels of SMAD2/3 were significantly increased upon SUSD2 knockdown. We next examined the expression of MKi67 and LGALS1 gene (the interaction partner for SUSD2).As a result, LGALS1 was significantly reduced upon SUSD2 knockdown.Moreover, MKi67 encoding the proliferation marker Ki-67 antigen, was significantly attenuated upon SUSD2 knockdown." -- -- -- -- Human L delay aging 28841682 Sen_G_0598 SPI1 6688 protein coding "BJ,WI-38" -- Acute leukemia Accelerate SA-β-gal activity assay//Western blot "The ectopic expression of Spi1 or HRASV12 both led to senescence that was characterized by stable cell cycle arrest, increased senescence-associated betagalactosidase (SA-βgal) activity, as measured by cytochemical staining and cytometric analyses, and increased protein levels of the senescence biomarker Dec1." -- -- -- -- p38 MAPK Activation GFP localization assay "The treatment of Spi1 overexpressing cells with SB203580 also increased, although partially, the number of GFP-positive cells, indicating that p38MAPK14 controls senescence and additional mechanisms modulating cell number." Human L delay aging 28912174 Sen_G_0599 IL4 3565 protein coding "CAKI-1,A498,786-O" -- Aging Accelerate Flow cytometry//Cell morphological analysis//SA-β-gal activity assay//Western blot//SAHF "Indeed, after exposure to IL-4 for 3–7 days, the cells underwent a profound morphological change characteristic of cellular senescence such as flattening and enlargement . Flow cytometric analysis showed that Caki-1 cells exposed to IL-4 for 7 days increased in size and granularity.Up-regulation of SA-β-gal activity increased protein expression of p21WAF1/CIP1 and p16INK4A and formation of senescenceassociated heterochromatin foci were also shown." STAT6//p38MAPK Activation//Activation Western blot "STAT6 phosphorylation was predominant by 10 min after IL-4 treatment.p38 MAPK as well as its upstream kinases, MEK3/6, were phosphorylated following IL-4 treatment." -- -- -- -- Human HL delay aging 23935100 Sen_G_0600 MAD2L1 4085 protein coding "IMR-90,MCF 10A" -- Aging Prevent SA-β-gal activity assay//Immunostaining//SAHF "Strikingly, several IMR90- siMAD2 cells were (SA)-β gal-positive (pH 6, 58%). The percentage of positive cells forgH2AX was higher (about 60%) in MCF10A-siMAD2 cells than in MCF10A-siGFP control cells .We then looked for SAHFs presence in primary human fibroblasts after 30 days from siMAD2 transfection and we scored that almost all of the cells (about 85%) showed heterochromatin foci when stained with DAPI." p14 -- Western blot//qRT-PCR "At 72 h post-transfection, p14ARF mRNA was highly increased in IMR90 human fibroblasts. Similarly, Western-blotting experiments showed a high increase of p14ARF protein levels in IMR90- siMAD2 cells, suggesting that p14ARF could mediate the senescence response in these cells by sensing aneuploidy." p53-p21 -- Western blot//qRT-PCR "Real-time RT-PCR revealed a high increase (sevenfold) of p21waf1 mRNA in primary human fibroblasts with MAD2 haploinsufficiency when compared to IMR90-siGFP control cells . As expected Western blotting confirmed that p21waf1 protein was significantly higher in siMAD2 than in IMR90-siGFP transfected cells. However, by Western blot analysis we detected elevated levels of p53 protein suggesting that the observed p21waf1 accumulation could be induced by p53 trans-activation of the p21waf1 gene. When p53 was posttranscriptionally silenced in IMR90-siMAD2 cells, expression level of the p21waf1 gene resulted similar to that present in IMR90-siGFP control cells, as assessed by real-time RT-PCR analysis." Human L delay aging 22170163 Sen_G_0601 SLC16A7 9194 protein coding "LoVo,HT29,HCT-8,HCT116,SW480 ,MKN45,MKN74" -- Colorectal cancer Prevent SA-β-gal activity assay//Cell morphological analysis//Immunostaining//SAHF "All the cancer cell lines examined, including those of the colon (HCT8, HCT116, HT29, LoVo, and SW480) and stomach (MKN45 and MKN74), displayed cellular enlargement and flattening as well as positivity for SA-β-Gal staining, following knockdown of MCT2. Cells additionally exhibited senescence-associated nuclear properties,including elevation of PML bodies, gH2AX, and SAHF." -- -- -- -- -- -- -- -- Human L delay aging 22964484 Sen_G_0602 FIS1 51024 protein coding Chang -- Aging Prevent SA-β-gal activity assay//Knockdown//Cell counting//XTT assay "The hFis1 knockdown cells started becoming progressively flattened on day 2 and were obviously flattened and enlarged on day 4, showing a phenotype similar to that of cells undergoing senescence. On days 4 and 5,~30% of the hFis1 RNAi cells stained positive for senescence-associated β-galactosidase , whereas no staining was observed in control cells. Microscopic evaluations revealed that the proliferation of hFis1-depleted cells was noticeably slow. Indeed, cell growth of the hFis1-depleted cells was found to be significantly retarded in measuring cell numbers and by XTT assays." OPA1 -- SA-β-gal activity assay//Knockdown//Flow cytometry//Immunostaining "Furthermore, hFis1 and OPA1 double-knockdown cells showed significant decreases in positive senescence-associated β-galactosidase staining as well as in cellular granularity.We found that prolonged depletion of hFis1 in fact increased ROS production on day 4. Moreover, sustained depletion of hFis1 caused a significant loss of mitochondrial membrane potential." -- -- -- -- Human L delay aging 17545159 Sen_G_0603 NFKB2 4791 protein coding A375 -- Melanoma Prevent Flow cytometry//SA-β-gal activity assay//Western blot//qRT-PCR "Cell size and granularity as determined by flow cytometry were significantly increased, as were the senescenceassociated β-galactosidase (SA-β-Gal) activity and HP1γ expression.The mRNA levels of p16 and p21 were increased after NF-kB2 silencing." EZH2//NIK Upregulation//-- qRT-PCR//Western blot "Using quantitative realtime PCR (qRT-PCR) in A375 melanoma cells, we observed a marked decrease in EZH2 mRNA under treatment with these NF-kB inhibitors.Similar to NF-kB2 silencing, NIK silencing strongly decreased the total NF-kB activity and abolished EZH2 promoter transcriptional activity. NF-kB2 overexpression (referred as o/e) in the melanocytes strongly increased EZH2 expression." -- -- -- -- Human L delay aging 26364600 Sen_G_0604 CAV1 857 protein coding MEF -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis "A higher percentage of caveolin-1-overexpressing MEFs (passage 1) are positive for acid β-galactosidase enzymatic activity as compared with MEFs derived from normal control mice.Light microscopy experiments indicated that 70±5% of MEFs(passage 1; n = 20) overexpressing caveolin-1 show a large and flat morphology as compared with 15±3% (n =18) of MEFs derived from normal control mice.In contrast, caveolin-1-overexpressing MEFs clearly showed premature irreversible growth arrest." -- -- -- -- -- -- -- -- Human L delay aging 12134086 Sen_G_0605 MIF 4282 protein coding MSC -- Aging Prevent Cell counting//MTT assay//qRT-PCR "The present study identified that 100 ng/ml MIF pretreatment in the presence of DOXO significantly increased MSC proliferation and cell viability compared with the DOXO group.Furthermore, MIF treatment significantly increased telomere length and restored telomerase activity, which were decreased by exposure to DOXO." -- -- -- -- PI3K-Akt Activation Western blot "Compared with the control group, treatment with DOXO at a concentration of 5 mol/l significantly decreased the phosphorylation of Akt, which was restored by MIF." Human L delay aging 29207187 Sen_G_0606 SIRT6 51548 protein coding "HuH-7,Hep3B" -- Hepatocellular carcinoma Prevent SA-β-gal activity assay//qRT-PCR//Cell morphological analysis "SIRT6-silenced cells were flattened and larger with the characteristic morphology of cellular senescence[17]. The percentages of SIRT6-depleted HCC cells positively stained with SA-β-gal were 29.1 4.9% (shSIRT6-1) and 16.2 8.4% (shSIRT6-3) Hep3B cells and 29.3 5.2% (shSIRT6-1) and 17.1 6.3% (shSIRT6-1) Huh-7 cells, while that of control HCC cells positively stained with SA-β-gal was less than 1.0% Hep3B cells and 6.1% Huh-7 cells. SIRT6 silencing increased mRNA levels of IL8, CXCL1, CXCL2 and CXCL3 ." -- -- -- -- -- -- -- -- Human HL delay aging 27824900 Sen_G_0607 CDK1 983 protein coding "RPE,U2OS" -- Aging Accelerate SA-β-gal activity assay//SAHF//Immunostaining "While long-term treatment with RO-3306 and NU6140 was toxic for cells, we found that all these markers were reduced upon combined Cdk1/2 RNAi or addition of Roscovitine, indicating that Cdk activity stimulates senescence." p21 Upregulation Western blot "p21 induction was reduced upon Cdk inhibition or depletion, suggesting that the remaining Cdk activity during a DDR promotes p21 expression. " -- -- -- -- Human L delay aging 28345297 Sen_G_0608 CDK2 1017 protein coding "RPE,U2OS" -- Aging Accelerate SA-β-gal activity assay//SAHF//Immunostaining "While long-term treatment with RO-3306 and NU6140 was toxic for cells, we found that all these markers were reduced upon combined Cdk1/2 RNAi or addition of Roscovitine, indicating that Cdk activity stimulates senescence." p21 Upregulation Western blot "p21 induction was reduced upon Cdk inhibition or depletion, suggesting that the remaining Cdk activity during a DDR promotes p21 expression. " -- -- -- -- Human L delay aging 28345297 Sen_G_0609 PRMT1 3276 protein coding "MCF-7,MDA-MB-231" -- Breast cancer Prevent SA-β-gal activity assay//qRT-PCR//Western blot//BrdU assay//Knockdown "The results revealed that both MDA-MB-231-shPRMT1 and MCF7-shPRMT1 cells showed a remarkably intensified SA-β-gal staining.Also, depletion of PRMT1 significantly increased the p21 expression, and reduced the CDC2, CCNB1 and CCNA2 expression at both mRNA and protein levels, in contrast to the control cells. Moreover, BrdU incorporation was remarkably diminished in PRMT1-depletion cells compared with control cells." ZEB1 Activation qRT-PCR//Western blot//CHIP "We found that only the ZEB1 expression was dramatically increased at both mRNA and protein levels, while the levels of other EMT inducers such as Twist, Snail and Slug, remained basically unchanged in MCF10A-PRMT1 cells. The results of quantitative chromatin immunoprecipitation (qChIP) assay revealed a prominent elevation in the enrichment of H4R3me2as, together with the presence of PRMT1, at the region between 327 and 179 bp of the ZEB1 promoter in MCF10A-PRMT1 cells." -- -- -- -- Human L delay aging 26813495 Sen_G_0610 KDR 3791 protein coding -- Colon Colitis associated cancer Prevent SA-β-gal activity assay//Western blot//Immunostaining "Specifically, senescence-associated beta-galactosidase (S-β-Gal) and the cyclin-dependent kinase inhibitor p16INK4A, were significantly upregulated in tumors of VEGFR2 IEC mice compared to controls.Altered tissue of VEGFR2?IEC mice revealed stronger expression of γH2A.X when compared to control mice using WB and IHC analysis." p21 -- Immunostaining//Western blot "We observed a significant difference in p21 protein expression in tumors of VEGFR2-deficient mice compared to control mice, as assessed by Western blotting and IHC.Moreover, not only the overall concentration of p21 was distinctively upregulated in tumors of VEGFR2 IEC mice, but also a predominant nuclear staining pattern could be observed." PI3K-Akt -- Co-IP "Using co-immunoprecipitation we were able to show an interaction of VEGFR2 and PI3K as well as AKT and p21, consistent with the idea of a functional connection of these molecules." Human L delay aging 25797700 Sen_G_0611 MYC 4609 protein coding -- "Liver,Lymphoma" Cancer Prevent SA-β-gal activity assay The suppression of MYC in primary MYC-induced lymphoma and hepatocellular carcinoma resulted in senescence-associated acidic β-gal (SA-β-Gal) staining. p15INK4b//p21CIP --//-- qRT-PCR "Among them, p15INK4b and p21CIP have been shown to be MYC targets.Upon MYC inactivation in vitro,osteosarcoma cell lines exhibited the induction of p15INK4b and p21CIP mRNA expression by quantitative RT-PCR. " -- -- -- -- Human L delay aging 17664422 Sen_G_0612 BRCA1 672 protein coding MEC -- Breast cancer Prevent SA-β-gal activity assay//Cell morphological analysis "We observed that Brca1MGKO MECs exhibited a large and flattened shape, a typical morphology of cellular senescence, while MECs from the other genotypes of mice were smaller and spindle-shaped. Notably, most Brca1MGKO MECs exhibited strong, peri-nuclear staining of SA-β-gal whereas only a small population of WT MECs and very few p16 -/- and p16 -/-;Brca1MGKO MECs showed positive staining." p16 -- Immunostaining//SA-β-gal activity assay//BrdU assay//Flow cytometry "We found that the percentages of Ki67-positive MECs in p16 -/- and p16 -/- ;Brca1MGKO mice (24.28±2.1 and 24.06±4.3) were significantly higher than those in WT mice (6.39±1.3), which in turn were significantly higher than the percentage in Brca1MGKO (2.20±0.2) mice. Notably, most Brca1MGKO MECs exhibited strong, peri-nuclear staining of SA-β-gal whereas only a small population of WT MECs and very few p16 -/- and p16 -/-;Brca1MGKO MECs showed positive staining. Importantly, MECs from p16 -/- and p16 -/-;Brca1MGKO mice displayed similar BrdU incorporation rates, 81% for p16-/-and 79% for p16-/-;Brca1MGKO, which were significantly higher than their WT counterparts. " -- -- -- -- Human L delay aging 27811360 Sen_G_0613 IGF1 3479 protein coding Aortic endothelial cell -- Atherosclerosis Prevent SA-β-gal activity assay "Incubation with 100 ng/mL IGF-1 for 24 h prior to hydrogen peroxide exposure significantly reduced expression of β-galactosidase activity, indicating that the enhanced antioxidant activity in response to IGF-1 counteracted oxidative stress induced premature cell senescence." GPX1 Upregulation Western blot//Enzyme activity assay//Immunostaining "Conversely，glutathione peroxidase (GPX) activity was upregulated in a dose-dependent and time-dependent manner.We further assessed GPX1 and GPX4 expressions by Western blot analysis. IGF-1 upregulated GPX1 (2.6-fold increase, 100 ng/mL IGF-1 vs 0 ng/mL IGF-1).Pre-exposure to IGF-1 dose-dependently suppressed ROS levels in both basal and oxidized LDL-stimulated cells. IGF-1's antioxidant effect was also time-dependent, since the ROS suppression by IGF-1 was more pronounced after 24 h exposure than at 1 h. " PI3K -- Western blot//GPX activity assay "The phosphatidylinositide 3-kinase (PI3k) inhibitor, LY294002, significantly suppressed IGF-1 upregulation of GPX1. IGF-1 upregulation of GPX activity was also significantly suppressed by LY294002 but not by PD98059 or SB202190, confirming that IGF-1 regulation of GPX was PI3k dependent." Human L delay aging 23261989 Sen_G_0614 TERT 7015 protein coding GBM -- Ataxia telangiectasia Prevent SA-β-gal activity assay//BrdU assay//TRF assay//Cell counting "The TRF assay showed that the telomere length of AT/TERT cells was extended to ＞12 kb, compared with ~4–11 kb for parental AT cells.Whereas parental AT cells underwent replicative senescence after ~24 PDs, AT/TERT cells survived for ＞70 to 100 PDs." -- -- -- -- -- -- -- -- Human L delay aging 14570874 Sen_G_0615 SOX5 6660 protein coding GBM Brain Glioblastoma Accelerate GO analysis//SA-β-gal activity assay "Interestingly, analysis of the genes upregulated by SOX5/6/21 instead resulted in a significant enrichment of GO-terms associated with general tumor suppressor responses, including Apoptotic process , Cellular response to stress Direct p53 effector and Senescence and Autophagy.Furthermore, forced expression of SOX5/6/21 significantly increased the number of cells that entered senescence in the three human primary GBM cell lines (KS4, G3 and #87), as measured by senescence associated gal-activity (SA-βgal) and the cleavage of X-gal." -- -- -- -- -- -- -- -- Human HL delay aging 28687615 Sen_G_0616 SOX6 55553 protein coding GBM Brain Glioblastoma Accelerate GO analysis//SA-β-gal activity assay "Interestingly, analysis of the genes upregulated by SOX5/6/21 instead resulted in a significant enrichment of GO-terms associated with general tumor suppressor responses, including Apoptotic process , Cellular response to stress Direct p53 effector and Senescence and Autophagy.Furthermore, forced expression of SOX5/6/21 significantly increased the number of cells that entered senescence in the three human primary GBM cell lines (KS4, G3 and #87), as measured by senescence associated gal-activity (SA-βgal) and the cleavage of X-gal." -- -- -- -- -- -- -- -- Human HL delay aging 28687615 Sen_G_0617 SOX21 11166 protein coding GBM Brain Glioblastoma Accelerate GO analysis//SA-β-gal activity assay "Interestingly, analysis of the genes upregulated by SOX5/6/21 instead resulted in a significant enrichment of GO-terms associated with general tumor suppressor responses, including Apoptotic process , Cellular response to stress Direct p53 effector and Senescence and Autophagy.Furthermore, forced expression of SOX5/6/21 significantly increased the number of cells that entered senescence in the three human primary GBM cell lines (KS4, G3 and #87), as measured by senescence associated gal-activity (SA-βgal) and the cleavage of X-gal." p53 Upregulation Western blot "Notably, despite a robust increase in p53 protein levels upon forced SOX21 expression, a corresponding upregulation of TP53 gene expression could not be detected." -- -- -- -- Human HL delay aging 28687615 Sen_G_0618 TP53 7157 protein coding PA-1(ATCC) -- Aging Prevent Western blot//SA-β-gal activity assay//Cell morphological analysis "We found that an increase in p16INKA4A expression was detected from days 4 5 post-treatment in TP53-silenced cells, corresponding with the partial release from G2 arrest and increase in (aberrant) mitoses.In control cells, ETO treatment evoked a modest but measurable increase in cell size (hypertrophy) and weak sa-β-gal staining at later time points. However, this was greatly accelerated by the silencing of TP53 with strong sa-β-gal staining in a significantly (p < 0.05, n = 3) higher proportion of cells in TP53-silenced (mean = 70.7%) vs. control (mean = 38.7%) samples at day 5 post-treatment." OCT4A//p21 --//-- Western blot "Transfection of PA-1 cells with TP53-siRNA successfully silenced TP53 expression throughout the time-course and, as expected, inhibited the increase in P21CIP1 in response to ETO treatment. However, unexpectedly, silencing of TP53 also largely inhibited the upregulation of OCT4A. To confirm this surprising finding, we repeated these experiments using PA-1 cells stably transfected with a shRNA vector directed against a different region of TP53 and saw the same results with diminished OCT4A upregulation." -- -- -- -- Human L delay aging 23287532 Sen_G_0619 AGT 183 protein coding VSMC Aorta Atherosclerosis Accelerate SA-β-gal activity assay "SA β-gal activity was significantly increased in Ang II–treated VSMCs compared with control cells.Consistent with our in vitro data, treatment with Ang II enhanced SA β-gal activity in the aortas of apoE-deficient mice." -- -- -- -- p53-p21 Upregulation Western blot "Moreover, expression of p21 and p53 was elevated in Ang II–treated VSMCs." Human L delay aging 16908765 Sen_G_0620 BAG3 9531 protein coding A172 -- Glioblastoma Prevent SA-β-gal activity assay//Cell morphological analysis//Cell viability assay//Colony formation assay//Flow cytometry//Knockdown "Of note, cells treated with BIS-specific siRNA (SiBIS) showed typical senescence-related phenotypic changes in a time-dependent manner: large, flattened morphology and gradually increased senescence-associated β-galactosidase (SA-β-Gal) staining: 86.8% of cells were positive for SA-β-Gal staining at 5 days after transfection. The proliferation rate was considerably slower in BIS knockdown cells compared in control cells as determined by relative increase in the cell numbers, 1.7-fold and 5.6-fold at day 5, respectively. The colony-forming ability was also prominently suppressed in SiBIS-treated cells, by 92% compared with control siRNA (SiCON)-treated cells. In addition, cell cycle profile demonstrated that the significant accumulation of cells in the G1 phase of the cell cycle accompanied by decrease in the cell populations in S or G2/M phase in SiBIS-treated cells, showing that the proportion of G1 phase was 84.7% and 59.9% in SiBIS- and SiCON-treated cells, respectively, at day 5." p27 -- Western blot//Knockdown The p27 levels were also progressively accumulated as increasing concentration of SiBIS. STAT3-SKP2-p27 -- Western blot//Knockdown "Immunoblottig showed that SKP2 levels were prominently decreased as BIS decreased, to 42% of control cells at day 5, which was inversely correlated with p27.We found that the phosphorylation of STAT3, representing the activated form of STAT3 as a transcriptional regulator, was profoundly decreased by BIS depletion in a time-dependent manner as determined by immunoblotting using specific antibodies for phospho-STAT3 (p-STAT3) that target pS727 and pY705.The depletion of STAT3 activation, accompanied with a decrease in Activate p-STAT3, both pY705 and pS727, downregulated SKP2 but increased p27 expression, comparable to what was observed following BIS depletion." Human L delay aging 25412315 Sen_G_0621 CDKN2A 1029 protein coding HCEC -- Aging Accelerate Immunostaining//qRT-PCR "Morphologically, the cells became flattened at Day 42 and floating at Day 49, suggesting an end replication and an end of cell life 20. During the period from Day 42 to Day 49 after the establishment of contact-inhibition in HCECs, there was continuous nuclear presence of p16INK4a and gradual upregulation of senescence markers such as ASF1A and GLB1." Bmi1 -- Knockdown//qRT-PCR "The same results were held up by Day 42 following weekly knockdown of p120-Kaiso, resulting in unique downregulation of p16INK4a transcript but upregulation of Bmi1 transcript ." -- -- -- -- Human L delay aging 27739458 Sen_G_0622 CTNND1 1500 protein coding HCEC -- Aging Prevent Knockdown//Immunostaining//qRT-PCR//SA-β-gal activity assay "In contrast,weekly knockdowns of p120-Kaiso siRNAs from Day 7 maintained the normal HCEC hexagonal morphology and density till Day 49 as reported11 and prohibited nuclear translocation of p16INK4a, expression of senescence markers,and increase of senescent cells during the entire experimental period." p16//Bmi1 --//-- Knockdown//qRT-PCR "The same results were held up by Day 42 following weekly knockdown of p120-Kaiso, resulting in unique downregulation of p16INK4a transcript but upregulation of Bmi1 transcript ." JAK2-STAT3//RhoA-ROCK --//-- Knockdown//qRT-PCR//Western blot "In contrast, 5 weekly knockdown of p120-Kaiso since Day 7 dramatically activated not only RhoA as reported11 but also promoted a sustained transcript expression of JAK2 and STAT3." Human L delay aging 27739458 Sen_G_0623 ZBTB33 10009 protein coding HCEC -- Aging Prevent Knockdown//Immunostaining//qRT-PCR//SA-β-gal activity assay "In contrast,weekly knockdowns of p120-Kaiso siRNAs from Day 7 maintained the normal HCEC hexagonal morphology and density till Day 49 as reported11 and prohibited nuclear translocation of p16INK4a, expression of senescence markers,and increase of senescent cells during the entire experimental period." p16//Bmi1 --//-- Knockdown//qRT-PCR "The same results were held up by Day 42 following weekly knockdown of p120-Kaiso, resulting in unique downregulation of p16INK4a transcript but upregulation of Bmi1 transcript ." JAK2-STAT3//RhoA-ROCK --//-- Knockdown//qRT-PCR//Western blot "In contrast, 5 weekly knockdown of p120-Kaiso since Day 7 dramatically activated not only RhoA as reported11 but also promoted a sustained transcript expression of JAK2 and STAT3 ." Human L delay aging 27739458 Sen_G_0624 PRMT6 55170 protein coding "MCF-7,MDA-MB-231" -- Breast cancer Prevent Knockdown//SA-β-gal activity assay//Cell morphological analysis//Cell counting "PRMT6 KD in both cell lines inhibits growth and specifically leads to a reduction of cells in S phase.Both sh-1 and sh-2 cells show a clear change in morphology, with cells looking bigger and flatter as compared with the scrambled control shRNA (sh-c) . As the morphology and the cell cycle arrest was reminiscent of cellular senescence, we confirmed this by SABG on the PRMT6 KD and control cells." p21 Downregulation qRT-PCR//Western blot//CHIP "Although p16 levels did not change in either MCF7 [the p16 locus is deleted in these cells (24)] or MDA-MB-231 (wt p16), we observed a striking upregulation of p21 messenger RNA (mRNA) and protein levels. In MCF7 cells, the p21 promoter is bound by PRMT6, methylated on H3R2me2a and H3K27me3, and p21 is expressed at low levels. " -- -- -- -- Human HL delay aging 22987071 Sen_G_0625 MYBL2 4605 protein coding HAEC -- Aging Prevent SA-β-gal activity assay//Cell morphological analysis "Importantly, the downregulated B-myb cells showed morphological change including a large-flat shape binucleation and polyploidy after 7 days. B-myb-silenced cells also displayed an increase in the proportion of SA-β-gal-positive cells compared with the control." -- -- -- -- ROS-p53-p21 -- Western blot//SA-β-gal activity assay//Immunostaining "Interestingly, the protein levels of p53 and p21 were significantly increased in B-myb-silenced cells compared with that of the controls.The upregulation of p- p53, p53 and p21 in B- myb knockdown cells could be abolished in the presence of PFTα. PFTα could also obviously attenuate the upregulation of SA- β- gal activity in B- myb knockdown cells. The production of intracellular ROS was significantly increased in B- myb- silenced cells.Bmyb- silenced cells incubated with NAC could significantly diminish the upregulation of p- p53, p53 and p21." Human L delay aging 27878894 Sen_G_0626 KLF6 1316 protein coding NIH-3T3 -- Aging Accelerate SA-β-gal activity assay The quantification of SA-β-Gal positive cells showed that the ectopic expression of KLF6 was able to increase the cellular senescence index. -- -- -- -- -- -- -- -- Human L delay aging 31824948 Sen_G_0627 SENP1 29843 protein coding HFF Skin Aging Prevent BrdU assay//SA-β-gal activity assay//Cell morphological analysis//Flow cytometry "In addition, shSenp1 caused substantial inhibition of cell proliferation.Cell cycle analysis showed that the fraction of HFFs in the G1 phase of the cell cycle increased from 53% in mock-infected controls to 76% in cells 5 days after infection with Senp1 shRNA.Cells infected with Senp1 shRNAs also exhibited an enlarged, flattened morphology characteristic of HFFs that have undergone replicative senescence.Greater than 90% of HFFs exposed to any of the three Senp1 shRNAs (#2, 3, and 8) exhibited blue-green cytoplasmic staining indicative of SA-βgal activity, whereas the vast majority of mock-infected HFFs or HFFs expressing the scrambled control shRNA were negative for S-Aβgal activity and exhibited normal morphology.Repression of Senp1 induced a gradual increase in cellular autofluorescence compared to shControlinfected HFFs, reaching approximately sixfold at 10 days after shRNA infection." -- -- -- -- p53 -- qRT-PCR "Although there was no significant change in levels of p53 mRNA, repression of Senp1 resulted in enhanced transcriptional activity of p53." Human L delay aging 18616636 Sen_G_0628 CDK2 1017 protein coding BJ -- Aging Prevent Immunostaining//SA-β-gal activity assay//Western blot "Indeed, in both human BJ fibroblasts and in MEFs, constitutive ectopic expression of CDK2 abrogated senescence in response to either DNA damage (irradiation), or oncogenic stress (HRasV12 transduction), as evidenced by continued proliferation by day 14 and absence of SABG staining,along with reduction in other protein markers of senescence." p53//Rb --//-- Western blot//qPCR "The lack of repression of CDK2 in p53-null cells and tissues suggested that CDK2 down-regulation is a downstream consequence of p53 activation.As expected, genetic ablation of Rb resulted in increased baseline protein levels of Cdk2." -- -- -- -- Human L delay aging 25149358 Sen_G_0629 HMGA1 3159 protein coding IMR-90 -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis//Western blot//SAHF "Cells expressing high levels of either HMGA protein underwent an acute cell cycle arrest and displayed features of senescence, including increases in p16INK4a and p53, a senescent morphology, and elevated SA-β-galactosidase activity.These cells also acquired SAHF-like foci that contained HMGA and me-K9H3." p16 -- Western blot "HMGA1-induced arrest and SAHF formation were clearly impaired in cells expressing sh-p16, despite having similar levels of transgene expression. " -- -- -- -- Human HL delay aging 16901784 Sen_G_0630 TERT 7015 protein coding mTert?/?Fibroblast -- Aging Prevent Western blot "In mTert-/- cells, the levels of the senescence biomarkers p53 and p21 were increased sharply at the M stage, in response to critically shortened telomeres at the same stage. However, unlike mTert -/- cells, the activation of the p53/p21 pathway in mTert+/+ cells immediately triggered the DDR response and up-regulated the expression of ATM, PARP1, and CHK1 at the M stage, and their elevated expressions were maintained through the L stage." -- -- -- -- -- -- -- -- Human HL delay aging 31481614 Sen_G_0631 PROM1 8842 protein coding CD1331 RPC -- Acute kidney injury Prevent SA-β-gal activity assay "The number of β-galactosidase expressing cells undergoing senescence appeared significantly higher in the CD133-Kd cells compared to their CD1331 control.The evaluation of T/S ratio revealed a significant reduction in telomere length in CD133-Kd cells compared to their CD1331controls,suggesting an implication of CD133 in the prevention of cell senescence." -- -- -- -- Wnt-β-catenin Activation qRT-PCR//Western blot//Luciferase reporter assay "Interestingly, CD133-Kd cell lines showed a reduced expression of Wnt4 after cisplatin-induced damage in respect to GFP cells. In addition, CD133-Kd cells showed an impaired response to the pharmacological activation of Wnt signaling pathway using CHIR99021 [31].Luciferase activity in CD1331 RPCs was significantly higher than the one in CD133-Kd cell lines, indicating a reduced β-catenin activation in CD133-Kd cells. Western blot analysis after immunoprecipitation of E-cadherin showed the concomitant presence of CD133 and b-catenin, suggesting that CD133 and E-cadherin may form a complex at the membrane level with β-catenin, thus limiting its cytoplasmic degradation. Indeed, β-catenin levels were significantly lower in CD133-Kd cells in respect to CD1331 RPCs both in basal culture conditions and upon stimulation with b-catenin stabilizer CHIR99021 .Indeed, β-catenin levels were significantly lower in CD133-Kd cells in respect to CD1331 RPCs both in basal culture conditions and upon stimulation with b-catenin stabilizer CHIR99021." Human HL delay aging 29431914 Sen_G_0632 STAG2 10735 protein coding BJ-2 -- Cancer Accelerate Immunostaining//SA-β-gal activity assay We observed a significant decrease in the levels of H2AX/p53BP1 DNA damage foci in SA2-depleted late PD cells.We observed a significant decrease in β-gal positive cells in SA2-depleted late PD cells. -- -- -- -- -- -- -- -- Human L delay aging 28819029 Sen_G_0633 CHEK1 1111 protein coding Fibroblast Foreskin Aging Accelerate Cell counting//BrdU assay "Numbers of Chk1 siRNA- and FANCD2 siRNA-transfected cells decreased equally until day 3 after irradiation.Forty-eight hours after siRNA transfection, fibroblasts were incubated with BrdU for 24 h. Four per cent of the control siRNA-treated fraction stained positive for the nucleotide analogue. Chk1 depletion induced BrdU incorporation in 15% of the cells." -- -- -- -- -- -- -- -- Human L delay aging 21995812 Sen_G_0634 FANCD2 2177 protein coding Fibroblast Foreskin Aging Accelerate Cell counting//BrdU assay//Knockdown "Numbers of Chk1 siRNA- and FANCD2 siRNA-transfected cells decreased equally until day 3 after irradiation.Forty-eight hours after siRNA transfection, fibroblasts were incubated with BrdU for 24 h. Four percent of the control siRNA-treated fraction stained positive for the nucleotide analogue.Remarkably, only 9% of the FANCD2 siRNA- transfected fibroblasts were BrdU-positive." -- -- -- -- -- -- -- -- Human L delay aging 21995812 Sen_G_0635 FBXO5 26271 protein coding RPE -- Aging Prevent Cell morphological analysis//Knockdown//SA-β-gal activity assay//BrdU assay "Cells lacking Emi1 were large and flattened and contained relatively large nuclei, a cellular morphology remi- niscent of cellular senescence.Approximately 37 to 65% of RPE cells stained positive for SA-β-gal at 4 or 5 days after Emi1 siRNA treatment, a phenotype that was recapitulated using two additional siRNA targeting sequences.We extended this analysis and detected a significant reduction in the prolif- erative capacity of RPE cells treated with human Emi1 siRNA." APC/C//E2F//Cyclin E1//ATM --//--//--//-- Knockdown//Cell morphological analysis//SA-β-gal activity assay//Western blot "Importantly, senescence induction upon Emi1 depletion required APC/C activity, as it was rescued upon codepletion of Cdh1, and was also observed in HCT116 and HeLa cells.Taken together, Emi1 knockdown results in APC/C activation and a consequent decrease in E2F target protein expression with the exception of an increase in cyclin E1 protein levels.A pronounced decrease in the percentage of senescent cells was measured when cells were grown for three days in the presence of KU-55933 before SA-β-gal staining (25% versus 75% in dimethyl sulfoxide-treated cells).In addition, ATM inhibitor-treated cells displayed a change in morphology from round and flattened (senescent-like) to more elongated and spindle shaped." -- -- -- -- Human HL delay aging 17875940 Sen_G_0636 RB1 5925 protein coding MSC -- Aging Prevent MTT assay//Alizarin red S staining//Western blot//qRT-PCR//SA-β-gal activity assay "Overexpression of RB in late-passage MSCs increased the proliferation rate and differentiation potentials; decreased the expression of senescence-associated markers such as p21, p16, and APO-1; and reduced β-gal staining." DNMT//c-JUN Upregulation//Binding Western blot "As expected, overexpression of RB in late-passage MSCs increased the protein levels of DNMT1.We found that c-JUN knockdown reduced DNMT1 expression in early- passage MSCs, but not late-passage MSCs, although the expression of RB in early-passage MSCs was not suppressed." -- -- -- -- Human L delay aging 25455074 Sen_G_0637 CIP2A 57650 protein coding AGS -- Gastric cancer Prevent SA-β-gal activity assay//Knockdown "In control AGS cells, less than 5% of them were β-Gal-positive, whereas up to 30% of CIP2A siRNA-treated cells were stained with β-Gal." -- -- -- -- -- -- -- -- Human L delay aging 18559589 Sen_G_0638 IL1B 3553 protein coding HUVEC -- Aging Accelerate SA-β-gal activity assay//Western blot "To address this, primary HUVECs were treated with recombinant human IL-1β for 6 days, the cell senescence was confirmed by SA-β-galactosidase activity.Furthermore, protein expression levels of senescence markers, such as p53 and p21, were also significantly upregulated in a time dependent way after IL-1β treatment with peak values of 2.8 fold and 3.1fold respectively." Caspase-1//NLRP3 --//-- Western blot "Interestingly, in the presence of z-YVAD-fmk, a caspase-1 inhibitor, mature IL-1 processing was abolished in senescent HUVECs but pro-IL-1 expression remained unchanged .SiNLRP3 or siASC transfection in the cells could significantly decrease their protein expression induced by bleomycin. As expected, IL-1 productions were also strongly attenuated in these silent senescent cells, but there was no significant effect in pro-IL-1 expression. " -- -- -- -- Human L delay aging 28064010 Sen_G_0639 SATB1 6304 protein coding MEF -- Breast cancer Accelerate SA-β-gal activity assay//Cell morphological analysis//Flow cytometry "MEFs expressing SATB1 grew markedly slower than controls,stained positive for β-galactosidase activity and had a flat morphology,indicating a large fraction of SATB1-expressing cells displayed signs of senescence.Consistent with this, we observed a large G1/G0 and a diminished S/G2 population." p16 Upregulation SA-β-gal activity assay//Western blot//Flow cytometry "In contrast to wild-type MEFs, SATB1 expression did not cause growth inhibition in p16-/-MEFs. Proliferation was comparable between SATB1- and control virus-infected p16-/- MEFs, and no change in senescence markers or cell cycle distribution was observed." Rb-E2F1 -- SA-β-gal activity assay//Co-IP "ATB1-expressing TM MEFs infected with RB virus had a markedly higher fraction of senescent cells compared with TM MEFs without SATB1. Both RB-HA and E2F1-HA were immunoprecipitated by Flag-tagged SATB1.The reciprocal interactions were also detected with HA-tagged E2F1 and RB, showing that SATB1 can interact with RB/E2F1 complexes." Human L delay aging 23686316 Sen_G_0640 PAK4 10298 protein coding MEF -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis "We found that Pak4-infected cells stained positive for SA-β-Gal activity.Interestingly, Pak4-expressing cells showed a large and flat shape." p16//p19ARF//Raf Upregulation//Upregulation//Activation SA-β-gal activity assay//Western blot "As expected, the control wild-type MEFs were readily arrested after infection with activated Pak4 or RasV12.However,when Pak4 was expressed in primary p19ARF/p16INK4a null MEFs ,the cells continued to grow and did not senesce.In addition，activated Pak4 led to a significant increase in p16INK4a and p19ARFlevels.Furthermore, Pak4 was able to induce phosphorylation of overexpressed and endogenous Raf on serine 338 in vivo, and it directly phosphorylated Raf on this site in vitro. In vitro kinase assays indicated that Pak4 could also stimulate Raf kinase activity." ERK-MAPK Activation Western blot//BrdU assay//SA-β-gal activity assay "We found that ERK activity was strongly stimulated in primary cells stably expressing Pak4 .?Furthermore, treating cells with a chemical inhibitor of the MEK-ERK pathway partially abrogated Pak4-induced premature arrest in primary fibroblasts and led to a decrease of about 50% in the number of SA-β-Gal-positive cells compared to what was seen in the absence of the drug." Human L delay aging 16227603 Sen_G_0641 CDC73 79577 protein coding "293T,A549,HUVEC" -- Cancer Prevent SA-β-gal activity assay//Knockdown//EdU assay "Higher percentage of positive SA-β-Gal staining cells was observed in all the three CDC73-KD cells. Moreover, reduced EdU incorporation and cell proliferation were detected upon depletion of CDC73 , indicating decreased expression of CDC73 can reduce DNA synthesis, which is also a well-known molecular phenotype of senescent cells." -- -- -- -- p53-p21 -- Western blot//Knockdown "However, both mRNA and protein levels of p53 and p21 were remarkably increased after CDC73 knockdown in 293T cells.Similarly, the protein level of p21 was significantly increased in CDC73-deficient HUVECs and A549 cells . These results support the notion that reduced CDC73 expression may induce senescence through the p53-p21 pathway." Human HL delay aging 29621547 Sen_G_0642 PRODH 5625 protein coding U2OS -- Aging Accelerate SA-β-gal activity assay//EdU assay "In accordance with our previous results, ectopic expression of PRODH modestly induced senescence in U2OS cells, as judged using SA-β-Gal and EdU assays.Furthermore, the substrate proline added to the medium enhanced the SA-β-Gal activation induced by PRODH overexpression in a dose-dependent manner, which suggests that PRODH induces senescence through the enzymatic activity." -- -- -- -- -- -- -- -- Human L delay aging 28264926 Sen_G_0643 tax 1491938 protein coding HeLa -- Aging Accelerate Western blot "We use the degradation of I-kBa as an indicator of NF-kB activation and the decrease in cyclin B1 and Skp2 levels with concomitant increase in the levels of p21 and p27 as surrogate markers for senescence induction.Wild-type Tax (WT) and those Tax mutants (V89A and M47) that are potent activators of NF-kB greatly reduced the levels of cyclin B and Skp2, and dramatically elevated p21 and p27 expression." RelA//HBZ --//-- Western blot//Knockdown//Luciferase reporter assay "Most interestingly, knockdown of RelA and to a rather limited extent, that of RelB, but not that of c-Rel or p100 prevented Tax-induced senescence.This is evidenced by the proliferation of EGFP+ HeLa-G/RelAKD cells and the absence of p21 and p27 up-regulation therein after Ad-Tax transduction.Like native HBZ, Flag-HBZ efficiently inhibits NF-kB activation by Tax." NF-κB Activation Western blot "Tax mutants that are impaired in NF-kB activation , in comparison, had only moderate or no effect on cyclin B, Skp2, p21, or p27." Human L delay aging 21552325 Sen_G_0644 SKP2 6502 protein coding MEF -- Prostate cancer Prevent SA-β-gal activity assay We also found that the ability of Ras to induce cellular senescence was far greater in Skp2-/- MEFs than in wild-type MEFs. p27//p21//ATF4 --//--//-- Western blot "Although p53 and p19Arf levels remained unchanged, we found that Skp2 deficiency cooperated with Pten inactivation or Arf loss to induce p27 expression. p21 expression was also increased in Pten+/- Skp2-/- and Arf-/- Skp2-/- MEFs.In contrast, Atf4 was markedly induced in Pten+/- Skp2-/- MEFs. Likewise, we also observed a marked increase in Atf4 protein levels, but not p-Perk, in Arf-/- Skp2-/- MEFs." -- -- -- -- Human L delay aging 20237562 Sen_G_0645 KCNJ12 3768 protein coding "PC-3,DU 145,LNCaP,MKN74,SNU638,SNU668,MCF-7,SK-BR-3,T47D" -- Cancer Prevent Cell morphological analysis//SA-β-gal activity assay//Knockdown//Western blot "Doxorubicin-treated PC-3 cells overexpressing Kir2.2 exhibited improved survival, decreased growth inhibition, and reduced accumulation of SA-β-Gal compared with doxorubicin-treated control PC-3 cells.All tested cancer cells from different tumor tissues, including prostate (PC-3, DU145, and LNCaP), stomach (MKN74, SNU638 and SNU668), and breast (MCF7, SK-BR3, and T47D),displayed cellular enlargement and flattening and were positive for SA-β-Gal staining following knockdown of Kir2.2, which lasted for up to 10 days .Previously reported senescence marker proteins,including PAI-1, osteonectin, and transglutaminase, were induced after Kir2.2 knockdown in the tested cancer cells." p27 -- Knockdown//Immunostaining//Western blot "Kir2.2 knockdown induced ROS accumulation. Rescue of Kir2.2 levels by Kir2.2 overexpression restored intracellular ROS to levels comparable with those in controls, indicating that the accumulation of ROS was directly de pendent on the level of Kir2.2.p27 knockdown decreased ROS generation and the ROS scavenger NAC prevented p27 synthesis in PC-3 cells after transfection of siKir2.2." -- -- -- -- Human L delay aging 20841375 Sen_G_0646 HRAS 3265 protein coding MEF -- Aging Accelerate Cell morphological analysis "In the case of overexpressed oncogenic HRas, cells were analyzed 6 days post-infection when, as expected, cells had a clear senescent morphology." -- -- -- -- -- -- -- -- Human L delay aging 19421407 Sen_G_0647 CDKN2A 1029 protein coding 2BS -- Aging Accelerate Cell morphological analysis//SA-β-gal activity assay//Flow cytometry "The 2BS/p16 cells showed increasing gross en- largement, flattening, and accumulation of granular cytoplas- mic inclusions.Only sporadic SA-β-gal-positive cells were seen in 2BS/ASp16 (A5, PD45) and young (PD29) cells, whereas almost all of 2BS/p16 (P3, PD43) cells were strongly stained, as were the senescent 2BS control cells (PD58).p16INK4acan inhibit the activity of CDKs, thereby blocking the entry of proliferating cells from G1to S phase." -- -- -- -- p16-CDK4-pRb -- Western blot "p16INK4acan inhibit the activity of CDKs,thereby blocking the entry of the proliferating cells to S phase through the p16INK4a/CDK4/cyclin D/pRB pathway. The function of p16INK4a is to maintain pRB in its hypophosphorylated Activate state.pRB phosphorylation status in 2BS/ASp16 (A10, PD50) cells (lane 1) is similar to that in young cells (PD25) (lane 2), which is considerably higher than that of senescent cells ." Human L delay aging 11606567 Sen_G_0648 SOX10 6663 protein coding "0002-ARM,UACC 1022,0380-MMU" -- Melanoma Prevent SA-β-gal activity assay//SAHF//Cell morphological analysis//Cell counting "Significant increases in SA-β-Gal activity were seen in all 3 lines when stably transduced with shSOX10 hairpins, suggesting that loss of SOX10 induces senescence in melanoma cells. Another hallmark of cellular senescence, senescence-associated heterochromatin foci (SAHF), was observed upon chromatin binding protein HP1b immunostaining .One week after infection, the shSOX10 lines showed reduced SOX10 protein levels and the cells became enlarged, flat, and translucent ). These cells also showed arrested or slowed growth." E2F1//Rb//MITF//p16//p27//p21 --//--//--//--//--//-- Western blot "In all three melanoma lines, SOX10 knockdown led to reduced levels of E2F1 protein and total RB protein.Reduced MITF protein was observed upon shSOX10 knockdown in both 0380-MMU and 0002-ARM cells.We observed moderate p16 expression in 0380-MMU cells;however,p16 levels remained unchanged following SOX10 knockdown. Weconsistently observed increases in p27 levels in all three cell lines as a result of SOX10 knockdown, similar to previous MITF knockdown studies. However, SOX10 knockdown caused an increase in p21 protein in all three-cell lines, thus showing a distinct difference from the MITF knockdown results." -- -- -- -- Human HL delay aging 23913827 Sen_G_0649 E2 1489080 protein coding HeLa -- Aging Accelerate SA-β-gal activity assay "After infection with the E2 virus, virtually all HeLa/LXSN cells displayed intense blue staining indicative of SA-β-gal activity and cellular senescence.The E2-infected HeLa/16E7 cultures contained cells displaying a flattened morphology and SAβ-gal activity interspersed with numerous proliferating colonies." E7 -- SA-β-gal activity assay//Cell morphological analysis "In contrast, as reported previously, the E2-infected HeLa/16E7 cultures contained cells displaying a flattened morphology and SA-β-gal activity interspersed with numerous proliferating colonies that did not stain, demonstrating partial protection from senescence by the wild-type HPV16 E7 protein." Rb Activation Western blot "The E2 protein repressed expression of cyclin A in HeLa/LXSN cells, indicating that the Rb pathway was activated." Human L delay aging 15126344 Sen_G_0650 SOX5 6660 protein coding Glial cell -- Glioma Accelerate Cell morphological analysis//SA-β-gal activity assay "We performed SA-β-gal staining of these cells and found that there were an increased number of SA-β-gal-positive cells in Ink4a-/- cells infected with Sox5+PDGFB or Sox5 alone.In Arf-/- cells, there was an increased number of SA-β-gal-stained cells in Sox5-infected cells only." p27//Akt Upregulation//Downregulation Western blot "When analyzing p27Kip1protein levels, we found that p27Kip1was clearly upregulated in the Sox5-infected Ink4a-/- cells compared with control and PDGFB-infected cells and that it remained high in the Sox5+PDGFB-infected cells.The total and activated levels of Akt were decreased specifically in the Sox5-stimulated Ink4a-/- cells compared with control and PDGFB cells and remained lower in the Sox5+PDGFB-infected cells compared with PDGFB alone." PDGFB -- Immunostaining "To study the proliferation status of Sox5 + PDGFB infected Ink4a-/- and Arf-/- cells, expression of Ki67, a marker of cell proliferation, was analyzed with triple immunofluorescence stainings . In Ink4a-/- cells, HA(PDGFB expression) and V5 (Sox5 expression) double-positive cells were completely negative for Ki-67, whereas in Arf-/- cells, about 20% of the HA + V5 double-positive cells also expressed Ki-67 ." Human HL delay aging 19219070 Sen_G_0651 G6PD 2539 protein coding HFF -- Aging Prevent SA-β-gal activity assay//Cell morphological analysis "There was an increase in the percentage of SA-β-Gal positive HFF1 cells, from less than 8% at PDL 22 to about 50% at PDL 52, and to over 95% at PDL 60. The majority of HFF3 cells remained SA-β-Gal negative at PDL 52, and a few cells (less than 5%) were stained positive at PDL 60.Some HFF1 cells showed the senescent morphology at PDL 22, and by PDL 60, nearly all the cells were senescen. By contrast, the HFF3 cells did not show such morphology even at passages up to PDL 60." -- -- -- -- -- -- -- -- Human L delay aging 10980404 Sen_G_0652 WIF1 11197 protein coding "LN-229,LN319" -- Glioblastoma Accelerate SA-β-gal activity assay//Cell morphological analysis//Immunostaining//Flow cytometry "We detected an increased percentage of senescence-like cells in WIF1-transfected clones with a positive relationship to WIF1 secretion of the respective clones. Similar differences were observed upon double staining of the cells with SA-β-galactosidase and DAPI, which visualizes nuclear morphology. In the LN319- and LN229-derived WIF1-overexpressing clones, we scored an increased population of both multinucleated and SA-β-galactosidase positive cells." -- -- -- -- Wnt Downregulation Luciferase reporter assay "Indeed, forced expression of WIF1 inhibited Wnt activity in a dose-dependent manner." Human HL delay aging 21642372 Sen_G_0653 CXCL8 3576 protein coding HUVEC -- Aging Prevent SA-β-gal activity assay//PI staining//Flow cytometry "However, IL-8 dose-dependently inhibited the increase of SA-beta-gal-positive cells when co-incubated with HUVECs.These results suggested that 150 μM H2O2 inhibited cell cycle progression at G1 phase, however IL-8 prevented this effect." hTERT Upregulation qRT-PCR QRT-PCR analysis showed that H2O2 down-regulated the expression of hTERT mRNA and the effect was suppressed by IL-8 in a dose- dependent manner. NF-κB//p38 MAPK Downregulation//Downregulation Western blot "H2O2 was found to activate NF-κB and p38(MAPK) pathway in a time-dependent manner, but the effect was suppressed by pretreatment of IL-8." Human L delay aging 23597430 Sen_G_0654 NAT10 55226 protein coding HGPS -- Hutchinson-Gilford progeria syndrome Accelerate SA-β-gal activity assay "Similarly, although NAT10 depletion strongly decreased the proportion of HGPS cells positive for senescenceassociated -galactosidase, this was not the case when NUP153 or TNPO1 was codepleted with NAT10." -- -- -- -- TNPO1 -- Knockdown//Microarray//Western blot "We observed that 70% of the genes showing significantly increased or decreased expression upon NAT10 depletion alone in control fibroblasts were not modified anymore upon simultaneous depletion of TNPO1, despite similar NAT10 protein knockdown." Human HL delay aging 29970603 Sen_G_0655 NUTF2 10204 protein coding "HGPS(HGPS 11513),Fibroblast" -- Hutchinson-Gilford progeria syndrome Prevent SA-β-gal activity assay//Knockdown "In addition to the effect on chromatin, disrupting nuclear Ran by depleting NTF2 also promoted the entry of control fibroblasts into senescence as measured by senescence-associated -galactosidase staining." -- -- -- -- -- -- -- -- Human HL delay aging 29970603 Sen_G_0656 OPA1 4976 protein coding -- "Liver,Skin,Colon" Aging Prevent SA-β-gal activity assay//Western blot "To further support the aging phenotype, we checked several markers of aging like β-galactosidase and p21. Both are significantly increased in liver, skin, and gut." -- -- -- -- -- -- -- -- Human L delay aging 28552492 Sen_G_0657 E2 1489080 protein coding HeLa/LXSN-1 -- Cervical cancer Accelerate SA-β-gal activity assay "After E2 expression, HeLa/LXSN-1 cells displayed a flattened morphology and intense blue staining indicative of SA-Gal activity and senescence." -- -- -- -- p53 Upregulation SA-β-gal activity assay//Western blot "Strikingly, the E2 protein did not induce the HeLa/16E7-p53CTF cells to display blue staining or senescent morphology;rather, the cells formed colonies that did not stain for SA-Gal activity.Full-length, wild-type p53 was expressed in all of the cell lines, and as expected, its expression was markedly induced by E2-mediated E6 repression." Human L delay aging 15047823 Sen_G_0658 SATB1 6304 protein coding -- Brain Parkinson's disease Prevent Western blot//SA-β-gal activity assay//Knockdown "Given this, we sought to investigate whether SATB1KO DA neurons present the classical features of cellular senescence. First we observed a dramatic increase in acidic lysosomal (SA-β-gal) activity, the hallmark senescence biomarker.We found upregulation of the majority of SASP factors at 50 days of differentiation in SATB1KO DA neurons versus WT neurons. We confirmed SASP activation by western blotting. In the conditioned medium of SATB1KO neurons, we found IGFBP7, which was absent in the medium of WT neurons." p21 Downregulation Western blot//CHIP//Knockdown//Luciferase reporter assay "Both CDKN1A transcription and p21 protein levels were significantly increased in SATB1KO DA neurons.Using ChIP-seq in DA neurons, we found that SATB1 binds the regulatory region of CDKN1A.To determine whether SATB1 directly represses CDKN1A, we performed a luciferase reporter assay and found that, under conditions of SATB1 knockdown, CDKN1A promoter activity was significantly increased ." -- -- -- -- Human HL delay aging 31543366 Sen_G_0659 SHH 6469 protein coding Human endometrial stem cell -- Recurrent pregnancy loss Prevent SA-β-gal activity assay//Western blot "Importantly, SHH significantly attenuated replicative senescenceinduced SA-β-Gal activity.The oxidative stress-induced expression of these senes- cence-associated (L-6, p16,p18, and p21) was significantly attenuated by SHH treatment. We conducted the additional set of experiments to further confirm the alleviating effects of SHH on oxidative stress- induced senescence with additional aging markers, such as RB1 and P14ARF. Consistently, oxidative stress-induced expression of these senescence-associated markers was significantly attenuated by SHH treatment." SERPINB2 Downregulation Western blot//qRT-PCR "Consistent with our hypothesis, replicative and oxidative stress-induced senescence significantly enhanced SERPINB2 expression at both the mRNA and protein levels.SHH treatment markedly suppressed SERPINB2 expression in endometrial stem cells. Importantly, senescence-induced SERPINB2 expression was significantly attenuated by SHH treatment at both the mRNA and protein levels." -- -- -- -- Human HL delay aging 31080015 Sen_G_0660 SPRY1 10252 protein coding ASC -- Aging Prevent Western blot//SA-β-gal activity assay//Knockdown//Growth curve assay//BrdU assay//ELISA "SPRY1 KO ASCs showed a proliferation arrest,CRISPR-GFP expressing cells served as negative control. Moreover, the anti-proliferative effect observed in SPRY1 KO ASCs was associated with significantly increased numbers of senescence-associated (SA) β-Galactosidase (β-Gal) positive ASCs and decreased Ki67 abundancy.CCL1 was not detectable by ELISAs, IL-8 and CXCL1/GROα, both are signature cytokines of senescent cells, were significantly elevated in SPRY1 KO ASCs compared to control cells." p53//p21//Rb --//--//-- Western blot SPRY1 KO ASCs showed a considerable upregulation of p53 and its target p21Cip1 associated with Rb activation (hypo-phosphorylation). Ras-MAPK//NF-κB --//-- Knockdown//Western blot Sprouty1 protein was depleted by both shRNA #1 and shRNA #2 and silencing of Sprouty1 elevated the abundance of phosphorylated ERK1/2 in PM4-stimulated ASCs compared to shCtrl indicating augmented MAPK activation.C/EBPβ protein was significantly elevated in SPRY1 KO ASCs and nuclear translocation of the NF-κB subunit p65 was significantly increased in these cells indicating an Activate NF-κB pathway. Human HL delay aging 32304210 Sen_G_0661 SRSF3 6428 protein coding "293T,HUVEC,MEF,NIH-3T3" -- Aging Prevent SA-β-gal activity assay//Knockdown//CCK-8 assay//Flow cytometry//qRT-PCR "RNA interferences using two shRNAs targeting SRSF3 caused increased senescenceassociated β-galactosidase (SA-β-gal) staining in both human (293T and HUVEC) and mouse (MEF and NIH3T3) cells. In addition, SRSF3-KD reduced cell growth rate in tested cell lines.Notably, SRSF3-KD caused a common decrease of S phase percentage in both 293T and MEF cells. What’s more,knockdown of SRSF3 in human and mouse cells also led to the decreased expression of MKI67, a molecular marker for cell proliferation." PTEN -- Luciferase reporter assay//Western blot "As knockdown of SRSF3 induced 3′ UTR shortening of PTEN and transcripts with shortened 3′ UTR of PTEN generated more protein than those with the longer one, one would expect that SRSF3-KD can increase the protein level of PTEN.Consistently, SRSF3 knockdown with two shRNAs both led to higher PTEN protein abundance in human 293T and HUVEC cells . " PI3K-Akt -- Knockdown//Western blot "Furthermore, SRSF3 knockdown attenuated the abundance of p-AKT but not that of the total AKT,coinciding with the result of PTEN upregulation." Human HL delay aging 30835716 Sen_G_0662 ITGB4 3691 protein coding Mice airway epithelial cell Mice airway epithelia Asthma Prevent SA-β-gal activity assay//Immunostaining//Knockdown Our results showed that ITGB4 deficient airway epithelial cells presented typical premature senescence. The rate of positive SA-β-gal staining in airway epithelial cells of ITGB4-/- mice was significantly higher than that in control mice.The cellular proliferative capacity of the airway epithelial cells in ITGB4-/- mice was decreased obviously compared with control mice. -- -- -- -- p53-p21 -- Knockdown//qRT-PCR//Western blot "Also, we have previously found increased expression of p53 and p21 in airway epithelia of ITGB4-/- mice. Therefore, we further detect the activation of p53/p21 signaling pathway by qPCR and western blotting in HBE cells. We found that p-p53 (the activated form of p53), p53 and p21 were significantly upregulated in ITGB4-silenced HBE cells. The upregulation of p-p53 was the most pronounced." Human L delay aging 30636108 Sen_G_0663 TRPC7 57113 protein coding -- Skin Aging Accelerate SA-β-gal activity assay//Knockdown//Western blot "In the absence of UVB, skin phenotype was similar between wild-type and TRPC7+/? or TRPC7?/? mice. In wild-type mice, UVB exposure induced severe desquamation and erythema of the skin. However, in TRPC7+/? and TRPC7?/? mice, UVB exposure induced slight or no damage and less epidermal thickening than in wild-type mice.SA-β-gal staining, used to analyze cell senescence, was also lower in the epidermis of TRPC7+/? and TRPC7?/? mice than in that of wild-type mice after UVB exposure.We found that after UVB irradiation, the knockdown of TRPC7 in keratinocytes significantly decreased the percentage of senescent cells,as determined by SA-β-gal staining,and improved the ratio of keratinocyte survival when compared with control cells. Furthermore, the knockdown of TRPC7 inhibited UVB-induced p53 expression in keratinocytes, which was corroborated by our observation that the UVB-induced activation of the p53 pathway was reduced in TRPC7+/? and TRPC7?/? mice." -- -- -- -- -- -- -- -- Human HL delay aging 31755176 Sen_G_0664 TET1 80312 protein coding "H1299,H1975,H226,H441,Calu-6" -- Lung cancer Prevent SA-β-gal activity assay//Cell morphological analysis//Colony formation assay//Crystal violet assay//Knockdown "TET1 knockdown significantly reduced proliferation of H1299, H1975, H226 and H441 cells at 120 h post-transfection by 63%, 40%, 24% and 21%, respectively, as determined using crystal violet assay. Furthermore, TET1 knockdown reduced anchorage independent growth of H1299, H226, and Calu6 lines as evident by 64%, 62% and 61% reduction in colony formation in soft agar, respectively.Cell morphology (enlarged size and flattened shape) suggested that these cells were undergoing cellular senescence and this was confirmed by analyzing the cells for β-galactosidase activity, a metabolic marker of senescence. Less than 1% of control cells showed detectable activity of this enzyme, while in the TET1 knockdown population, approximately 40% (H1299), 18% (H1975) and 6% (H226) of cells were β-galactosidase positive." p53//p21 Binding//-- CHIP//qRT-PCR "As expected, no enrichment of p53 at its predicted binding site within the TET1 promoter region was detected in H1299 cells characterized by a p53-null mutation. Conversely, 23,800- and 13,500-fold enrichments of p53 at the TET1 promoter were found in the WT p53 cell lines A549 and H2228,respectively. Furthermore, p53 binding to the TET1 promoter was reduced ≥90% in p53-mutated cell lines (enrichment of 1,600-, 1,200-, 223- and 16- fold in H1975, Calu6, H1993 and SW900, respectively) and negatively correlated with overall TET1 expression.qRT-PCR analysis demonstrated that expression of p21 is elevated 6- to 70- fold in H1299 cells in a highly synergistic and dose-dependent manner by combined TET1 knockdown and chemotherapy treatment." -- -- -- -- Human HL delay aging 30622117 Sen_G_0665 SARS1 6301 protein coding "HeLa 1.2.11,BJ" -- Aging Accelerate SA-β-gal activity assay//Knockdown//FISH "We observed an unexpected increase in SA-β-gal activity and increased levels of cellular senescence signaling molecules, such as P21, P16, and β-galactosidase (β-Gal)32–34 at late cell passages of both cells; these changes were even observed in HeLa 1.2.11 cells, which undergo little replicative senescence, suggesting a role of SerRS in promoting cellular senescence beyond its role in translation.Significant telomere shortening was viewed by reduced FISH signals, further indicating that telomeres were globally shortened when SerRS was overexpressed." POT1 Binding Pull-down assay//Immunostaining//CHIP//Co-IP "SerRS was partially colocalized with POT1 in the nucleus.The interaction between SerRS and POT1 was further confirmed by their Co-IP from HeLa cells.V5-tagged POT1 was able to be coprecipitated with Flag-tagged SerRS via Flag antibody-mediated isolation; the reverse experiment with a V5 antibody produced a complementary result. In HeLa VST cells, the endogenous SerRS proteins could be coprecipitated with endogenous POT1 by POT1 antibody.The GST pull-down assay clearly showed the direct interaction between SerRS and POT1." -- -- -- -- Human L delay aging 31815007 Sen_G_0666 TLR4 7099 protein coding MLEC -- Emphysema and COPD Prevent SA-β-gal activity assay//Knockdown//Flow cytometry "TLR4?/? mouse lung Ec (MLEC) showed increase in SA‐β‐gal activity, even at low passages, compared to WT MLEC.To further investigate the role of TLR4 in cellular senescence, we examined the cell growth rate and cell cycle analysis between TLR4?/? and WT MLEC. TLR4?/? MLEC had a significantly decreased rate of cell growth compared with WT MLEC. Analysis of cell cycle distribution by flow cytometry showed that TLR4?/? MLEC exhibited increased G1 phase (83.4 ± 1.4% vs. 74.5 ± 3.8%) and decreased G2 phase (10.0 ± 1.5% vs. 16.8 ± 2.4%) compared to WT MLEC." p16//HDAC2 Downregulation//-- qRT-PCR//Western blot "In contrast, TLR4 overexpression resulted in a significant decrease in both p16INK4a mRNA and protein expression in hLMVEC.We found that HDAC2 mRNA expression significantly decreased in lungs from TLR4?/? mice compared to WT. We also found that phosphorylated (p)and total‐HDAC2 protein expression were significantly decreased in TLR4?/? MLEC compared to WT.As further confirmation, HDAC2 siRNA sig- nificantly increased p16INK4a." -- -- -- -- Human L delay aging 30790400 Sen_G_0667 MANF 7873 protein coding -- Adipose Aging Prevent SA-β-gal activity assay//Knockdown//qRT-PCR "We also found signs of cellular senescence in MANFHet mice, but not WT littermates, at 10 months of age: elevated senescence-associated βGal activity in subcutaneous and visceral WAT, increased levels of 4-HNE adducts, and increased expression of p16 and IL-6." -- -- -- -- -- -- -- -- Human HL delay aging 31489403 Sen_G_0668 CD9 928 protein coding HUVEC -- Atherosclerosis Accelerate Cell morphological analysis//BrdU assay//Flow cytometry//Immunostaining//SA-β-gal activity assay//Western blot "Young cells transduced with CD9 adenovirus were enlarged and flattened. Moreover, CD9 upregulation increased SAβG staining, but decreased BrdU incorporation, Ki67 immunoreactivity, the proportion of cells in the S phase, and endothelial tube formation. In addition, the levels of p53, p-p53, and p21 proteins increased." -- -- -- -- PI3K-Akt-mTOR-p53 Activation Knockdown//Western blot//SA-β-gal activity assay "Pretreatment with LY294002, a specific inhibitor of PI3K [42], or rapamycin, an inhibitor of mTOR [43], reduced the levels of p53 and p21 and SAβG staining induced by CD9 overexpression. Knockdown of PIK3CA, but not that of PIK3CB, significantly decreased the levels of pAKT, p53, p-p53, pS6K, and p21 proteins, as well as SAβG staining, suggesting that PIK3CA might be involved in CD9- induced cellular senescence. These results indicate that the PI3K-AKT-mTOR-p53 pathway might regulate CD9- mediated cellular senescence." Human HL delay aging 32346137 Sen_G_0669 PRDX6 9588 protein coding C2C12 Gastrocnemius Metabolic Sarcopenia Prevent SA-β-gal activity assay//Knockdown//qRT-PCR "We screened the expression of 84 key genes involved in the senescence program on gastrocnemius of wt and Prdx6-/- mice. A significant up-regulation of p53 (p < 0.05),Cdkn1b (p < 0.05) and Cdkn1c (p < 0.05), was found in Prdx6-/- mice.Prdx6-/- mice showed increase in activity of SA-β-Gal compared to wt mice (p < 0.005).The expression levels of telomeric repeat-binding factor 1 (TERF1), telomeric repeat-binding factor 2-interacting protein 1 (TERF2IP), telomerase-associated protein 1 (TEP1), and regulator of telomere length 1 (RTEL1), were significantly reduced in Prdx6-/-mice compared to wt mice (p < 0.05)." SIRT1 -- Western blot//Knockdown "Following the nuclear and cytoplasmic proteins fractionation, indeed, we found that in Prdx6-/- mice SIRT1 localized predominantly in the cytoplasm (p < 0.05) rather than in the nucleus (p < 0.0005) compared to wt, suggesting reduced levels of Prdx6 lead to an impairment of SIRT1 nuclearcytoplasmic shuttling." p53-p21 -- Western blot//Knockdown "As expected, p53 acetylation was significantly increased in Prdx6-/- mice compared to wt mice, confirming the SIRT1 reduced nuclear activity. Acetylated p53 induces p21, a cyclin-dependent kinase inhibitor 1 able to regulate cell cycle and directly blunt telomerase activity . Consistently with the reduction of ATLR and increase in p53 activation, we observed a significantly enhanced steady-state levels of p21 in Prdx6-/- mice." Human HL delay aging 32316601 Sen_G_0670 BMI1 648 protein coding B-MSC -- Aging Prevent SA-β-gal activity assay//Knockdown//Cell counting//qRT-PCR "We then serially passaged BMSCs in vitro to examine the proliferative capacity of Bmi1-deficient BMSCs. Whereas wildtype BMSCs expanded significantly over three passages, Bmi1-deficient BMSCs ceased proliferation upon passage 2 and failed to expand in later passages.Bmi1-deficient BMSC cultures exhibited significantly more senescent-associated β-gal (SAβ-gal)+ cells and had increased expression of Ink4a, a marker of senescent cells." -- -- -- -- -- -- -- -- Human HL delay aging 31257132 Sen_G_0671 SIRT2 22933 protein coding NP -- Disc degenerative disease Prevent SA-β-gal activity assay//Western blot//Knockdown//RT-PCR//Flow cytometry "NP cells expressed significantly higher β-gal and MMP3/9 levels with higher IL-1b stimulation. NP cells were transfected with Lenti-sirt2 to overexpress Sirt2 protein. Meanwhile, Lenti-NC was used as the sham group. The function of lentivirus transfection was verified through WB analysis. Results indicated that these cells exhibited significantly less β-gal, MMP3/9 activity than those in IL-1b-induced group. Furthermore, IF staining, WB and RT-PCR analysis demonstrated that the expression of type II collagen was significantly up-regulated.Flow cytometry result showed that compared with the control group, more cells stayed in the G1 phase and less cells stayed in the M phase in the two IL-1b stimulation groups. Total ROS level increased remarkably under the treatment of IL-1b." IL-1β//SOD1//SOD2 Downregulation//Upregulation//Upregulation RT-PCR//Western blot//Flow cytometry " Results indicated that these cells exhibited significantly less β-gal, MMP3/9 activity than those in IL-1b-induced group.Sirt2 expression decreased significantly in cells after treatment with IL-1b. Meanwhile, total ROS level decreased obviously under the treatment of Lenti-sirt2.In addition, anti-oxidative SOD1 and SOD2 were remarkably up-regulated compared with IL-1b- induced group." p53-p21 Downregulation Western blot//RT-PCR Western blot and RT-PCR results indicated that Sirt6 overexpression significantly decreased the expressions of p53 and p21. Human L delay aging 30981502 Sen_G_0672 LSG1 55341 protein coding MRC-5 -- Aging Prevent SA-β-gal activity assay//Knockdown//BrdU assay//Western blot "Next, we used siRNA SMARTpools to EFL1 and LSG1 and observed the expected reduction in BrdU incorporation, induction of acidic β‐galactosidase activity and induction of p16 immunoreactivity." -- -- -- -- p53 -- Knockdown//BrdU assay//qRT-PCR//DAPI staining "E6（E6 abrogated p53 expression） expression led to maintained BrdU incorporation upon LSG1 knockdown, indicating p53 dependence. Expression of E7（E7 expression enhanced p53 levels）did not rescue the inhibition of cell cycle elicited by LSG1 knockdown. Once again, inhibition of the p53 pathway led to bypass of shLSG1‐induced proliferative arrest . Finally, we used shRNA to p53 to follow the growth characteristics of cell lines transduced with shRNA to LSG1. p53 knockdown resulted in greatly accelerated growth rates in vector control cells, and the knockdown of LSG1 failed to inhibit growth in these cells." Human HL delay aging 31148378 Sen_G_0673 SBDS 51119 protein coding MRC-5 -- Aging Prevent SA-β-gal activity assay//Knockdown//BrdU assay//Western blot "Next, we used siRNA SMARTpools to EFL1 and LSG1 and observed the expected reduction in BrdU incorporation, induction of acidic β‐galactosidase activity and induction of p16 immunoreactivity." -- -- -- -- p53 -- Knockdown//BrdU assay//qRT-PCR//DAPI staining "E6（E6 abrogated p53 expression） expression led to maintained BrdU incorporation upon LSG1 knockdown, indicating p53 dependence. Expression of E7（E7 expression enhanced p53 levels）did not rescue the inhibition of cell cycle elicited by LSG1 knockdown. Once again, inhibition of the p53 pathway led to bypass of shLSG1‐induced proliferative arrest . Finally, we used shRNA to p53 to follow the growth characteristics of cell lines transduced with shRNA to LSG1. p53 knockdown resulted in greatly accelerated growth rates in vector control cells, and the knockdown of LSG1 failed to inhibit growth in these cells." Human HL delay aging 31148378 Sen_G_0674 LMNB1 4001 protein coding HBEC -- Chronic obstructive pulmonary disease Prevent SA-β-gal activity assay//Knockdown//Immunostaining//ELISA//Western blot "Lamin B1 knockdown was sufficient to induce cellular senescence by means of SA-β-gal staining, phospho-histone H2A.X (Ser139) staining of DNA damage, and Western blotting of CDKN2A/p16 and CDKN1A/p21, which was significantly enhanced by CSE treatment in HBEC. To determine SASP status, CXCL8 secretion was examined. Significant increase in CXCL8 secretion was observed in conditioned medium only from CSEtreated HBEC with lamin B1 knockdown, indicating that both CSE and lamin B1 reduction to some extent are necessary for progression to full senescence with SASP." -- -- -- -- mTOR Downregulation Western blot//Knockdown//qRT-PCR "In comparison with non-smoker lungs, electron microscopic evaluations showed a significant increase in mitochondrial counts in airway epithelial cells in COPD lungs . In comparison with nonsmokers and non-COPD smokers, lamin B1 and DEPTOR mRNA were significantly reduced in HBEC of COPD patients. Consistent with CS-exposed mouse models, no apparent reduction of lamin B1 and DEPTOR expression levels and no increase in p-S6K were demonstrated in alveolar lesions in COPD lungs. These results suggest the existence of pathogenic link between reduced lamin B1–mediated MTOR signaling and enhanced mitochondrial accumulation associated with accelerated cellular senescence in airway epithelial cells during COPD development." Human L delay aging 30692212 Sen_G_0675 STUB1 10273 protein coding HEK293T -- Aging Prevent SA-β-gal activity assay//Western blot "The immunoblotting results showed that H2O2 increased the expression of p53 and p21 in HEK293T cells, indicating the occurrence of cell senescence. When HEK293T cells were transfected with STUB1 plasmid and treated with H2O2, cell senescence was significantly reduced, demonstrated by the decrease of SA-β-Gal positive cells and by the reduction of p53 and p21." BMAL1 Downregulation Western blot The endogenous BMAL1 protein was significantly reduced after expressing Myc-STUB1.Gradient increase of the transfected STUB1 led to a progressive reduction of BMAL1 . -- -- -- -- Human L delay aging 32041778 Sen_G_0676 BTK 695 protein coding -- Brain Aging Accelerate SA-β-gal activity assay//Knockdown//qRT-PCR "To test whether BTK inhibition could prevent the age-dependent buildup of senescent cells in the brain, we measured the expression of different markers of senescence in brains of control and ibrutinib-treated Zmpste24?/? mice.This was concomitant with a decrease in p53 and p16 levels, as expected. In line with this, mRNA levels of BTK (which is a transcriptional target of p53 (Althubiti et al., 2016)) and other markers of senescence were also decreased in the brain samples. p53 mRNA levels did not change significantly, which is compatible with the post-translational effects of BTK on p53 levels (Althubitiet al., 2016). The reduction in senescent cell accumulation in brains of treated mice was confirmed by a whole organ SA-β-gal staining." -- -- -- -- -- -- -- -- Human L delay aging 31736210 Sen_G_0677 CDKN2A 1029 protein coding AECII -- Chronic obstructive pulmonary disease Accelerate SA-β-gal activity assay//Knockdown//EdU assay "In vivo cell proliferation was assessed by EdU incorporation. Deleting p16 significantly increased cell proliferation from 3.6% to 5.3%.When exposed to CSE, the percentage of senescent p16+/+ AECIIs increased as assessed by flow β-gal activity. Deletion of p16 completely prevented the CSE-induced senescence of AECII (p16+/+ CS vs. p16?/? CS)." Cyclin D -- Western blot "There was a much larger increase in cyclin D in p16?/?compared with p16+/+lungs upon CS exposure, supporting the observation of enhanced cell proliferation and regeneration in p16?/?lung. " IGF-1-Akt1 -- qRT-PCR//Western blot//Knockdown "Deletion of p16 increased IGF1 mRNA and protein. This translated to a 7.83-fold increase in IGF1 protein in p16?/? lung compared with p16+/+.Similarly, total Akt was substantially elevated in p16?/? lungs and increased with CS." Human HL delay aging 31428695 Sen_G_0678 ATP6AP2 10159 protein coding C2C12 -- Sarcopenia Accelerate SA-β-gal activity assay//Western blot "In the differentiated myo-tube from (P)RR‐expressing C2C12 cells, we detected positive SA‐β‐gal staining with an increase in γH2AX, a senescent marker, and increased expression of TNF‐α, a senescence‐associated secretory phenotype, indicating that (P)RR expression induced cellular senescence. Moreover, aging markers, such as p16, p21, and γH2AX, were up‐regulated in the muscles of both (P)RR‐Tg mice and aged WT mice." -- -- -- -- Wnt-YAP Activation Western blot//Immunostaining "As expected, ABC was significantly increased and nuclear localization of β‐catenin could be observed in the muscles of (P)RR‐Tg mice, indicating activation of Wnt/β‐catenin signaling by (P)RR .Wnt and YAP activities were enhanced in myotubes from (P)RR‐expressing C2C12 cells. Treatment with verteporfin, a small molecule that inhibits the assembly of YAP/TEAD and its transcriptional activity, significantly restored myotube formation in (P)RR‐expressing myoblasts." Human L delay aging 31282603 Sen_G_0679 SIRT1 23411 protein coding HUVEC -- Heart failure Prevent SA-β-gal activity assay The inhibition of Sirt1 activity by EX-527 caused an increase of senescence in RP-ECs compared with baseline (P = 0.001). Cat//SOD --//-- Enzyme activity assay "The CRP enhanced Sirt1 activity measured in PBMCs from patients (RP vs P, P= 0.02). Likewise, Cat and SOD activities measured in serum were greater in RP than in P (P < 0.005 and P < 0.05, respectively)." -- -- -- -- Human L delay aging 28331525 Sen_G_0680 GDF11 10220 protein coding "HFL-1,Primary bronchial epithelial cell" -- Chronic obstructive pulmonary disease Prevent SA-β-gal activity assay//Western blot//Cell counting "Treatment with GDF11 significantly inhibited the CSE-augmented expression of p16 and p53 and the CSE-augmented SA-β-gal activity in a concentration-dependent manner. CSE significantly delayed cell proliferation, but treatment with GDF11 ameliorated the delay in cell growth. Similar anti-senescent effects of GDF11 were observed in primary bronchial epithelial cells. Treatment with GDF11 significantly ameliorated the CSE-induced cellular senescence as assessed by the expression of senescence-associated proteins, SA-β-gal activity and cell proliferation.These results suggest that GDF11 could prevent cigarette smoke from accelerating lung cellular senescence." -- -- -- -- Smad2/3 -- Western blot "As previously reported in other types of cells,GDF11 phosphorylated Smad2/3,and the phosphorylation was inhibited by an activin receptor-like kinase (ALK)4/5 inhibitor, SB431542, in HFL-1 cells." Human L delay aging 28455454 Sen_G_0681 SATB2 23314 protein coding B-MSC Bone Aging Prevent SA-β-gal activity assay//Immunofluorescence "To examine replicative senescence, we repeatedly subcultured T-BMSCs transfected with either Lev-Satb2 or Lev-GFP.We observed that T-BMSCs that overexpressed Satb2 exhibited enhanced anti-aging capacity, as revealed by SA-β-gal staining at the 10th passage.Compared with the Lev-GFP group, the Lev-Satb2 group displayed more intensive GFP staining in both the bone matrix and the surrounding fibroblastic-like tissue, indicating that more exogenous cells survived in the Lev-Satb2 group." -- -- -- -- -- -- -- -- Human HL delay aging 25200657 Sen_G_0682 NAMPT 10135 protein coding SMC -- Atherosclerosis Prevent SA-β-gal activity assay//Knockdown "In the longer lived HITC6 SMCs,lifespan extension by Nampt was even more striking, with an additional 6.3±0.3 population doublings or a 71 ±7% extension of lifespan.We quantified the proportion of senescent SMCs in successive subcultures incubated with 10 nM FK866, a concentration we determined reduced Nampt activity in SMCs to 22 ± 2% of baseline.Kaplan-Meier survival analysis revealed a substantially shortened senescence-free survival of Nampt-inhibited cells SMCs. In contrast, there was markedly extended senescence-free survival in Namptoverexpressing SMCs versus vector-infected cells." SIRT1//p53 Activation//Downregulation Western blot "SMCs overexpressing Nampt had a 86 ± 4% (p=0.03, n =4) increase in SIRT1 activity. We were surprised to find that Nampt overexpression also modestly increased abundance of SIRT1 protein (1.3±0.3-fold, p =0.02), although not enough to fully account for the increased deacetylase activity.this agerelated increase in p53 was blunted in parallel cultures of SMCs overexpressing Nampt. Furthermore, the fraction of p53 that was acetylated on Lys-382 was substantially lower in Nampt-overexpressing SMCs than in control cells. This p53 modification was associated with a significantly increased rate of p53 degradation in Nampt-overexpressing SMCs, as assessed in SMCs incubated with cycloheximide." -- -- -- -- Human L delay aging 17307730 Sen_G_0683 SATB2 23314 protein coding B-MSC -- Aging Prevent SA-β-gal activity assay//Knockdown "Although cellular proliferation remained largely unaltered, enforced SATB2 overexpression significantly enhanced pluripotency markers expression, whereas reduced cellular senescence as reflected by reduced SA-β-Gal-positive cells and senescence marker expression.The cellular proliferation rate significantly decreased after SATB2 knockdown." -- -- -- -- Nanog Upregulation Knockdown//qRT-PCR//CHIP "Our data reveal that the level of Nanog transcript was induced upon SATB2 overexpression, and reduced following SATB2 knockdown.Significant enrichment of SATB2 was identified in four putative binding sites in Nanog promoter region." Human L delay aging 27632702 Sen_G_0684 PRDX2 7001 protein coding Human primary Chondrocyte -- Osteoarthritis Prevent SA-β-gal activity assay//Western blot//RT-PCR//Immunostaining "Western blot analysis showed that IL-1β-induced chondrocytes expressed a large amount of β-gal, p53, and p21, while Prx II could attenuate the effect of IL-1β and decrease the expression of β-gal, p53, and p21. RT-PCR results showed that Prx II increased the expression of anti-aging molecule BMI-1 and decreased the expression of p53 and p21. Cellular immunofluorescence also showed that Prx II reduced the expression of β-gal induced by IL-1β." -- -- -- -- p16 Downregulation RT-PCR//Western blot "Western blot results showed that IL-1β-induced chondrocyte expressed more p16 while CDK4, CDK6, and E2F1 decreased, and overexpression of Prx II attenuated the effect of IL-1β. The results of RT-PCR were similar to those of Western blot." Human L delay aging 32329817 Sen_G_0685 MSN 4478 protein coding HDMEC -- Aging Prevent Cell morphological analysis//SA-β-gal activity assay//Knockdown//Western blot//RT-PCR "Moesin knock-down HDMECs showed the characteristics of aging, i.e., they were large, flat, and stellate, beginning at the early stage of passage 6. Moesin knock-down HDMECs from the early stage of passage 6 were stained blue with SA-β-gal. HDMECs at late passages higher than passage 25 stained blue in all serial subcultures. In comparison with control vector-treated HDMECs, the doubling time of moesin knock-down HDMECs was delayed by 3 h at passage 6 and 10 h at passage 22. The doubling level of the cumulative cell population in moesin-knockdown HDMECs was much less than that in virus-untreated HDMECs. Moesin knock-down HDMECs expressed less moesin RNA than control vectortreated HDMECs. In addition, the RNA expression of p16 in HDMECs at late passages and moesin knockdown HDMECs was higher than that in control vectortreated HDMECs.In applying Western blotting, reduced expression of moesin and higher expression of p16 were observed in moesin knock-down HDMECs, compared to control vectortreated HDMECs." -- -- -- -- -- -- -- -- Human L delay aging 20376899 Sen_G_0686 SGK1 6446 protein coding "HUVEC,SGK1WT" -- Atherosclerosis Prevent SA-β-gal activity assay//Telomerase activity assay "Infected HUVEC lines have been cultured until the last stage of senescence, represented as arrest of cellular proliferation (PDL16). During serial passages of HUVEC, the activity of SA-β-gal in control pLPCX and in SGK1Δ60 increased by ~5-6-fold from PDL6 to PDL16. SGK1WT was able to significantly reduce SA-β-gal activity (by 2-fold) compared with SGK1Δ60 and pLPCX cellular groups at PDL8 (p<0.0005). SGK1WT cells showed a significant increase in telomerase activity at PDL8 compared with both PDL6 (p<0.05), and PDL13 (p<0.005)." hTERT Binding Co-IP//Pull-down assay//Flow cytometry//Immunostaining "We found that SGK1 co-immunoprecipitates with hTERT in all cellular constructs, proving a protein-protein interaction. However, a different shift was observed between total SGK1Δ60 and hTERT immunoprecipitate, suggesting that hTERT interacts only with SGK1 full length,and further supporting the previously reported inactivity of SGK1Δ60 in delay endothelial aging. To confirm these results we performed a GST pull-down assay which established the direct interaction between SGK1 and hTERT, similarly to what we observed with previous used methodology.However, a significant decrease in ROS production was evident in the cells overexpressing SGK1WT compared with both control, and SGK1Δ60 construct (p<0.005)." -- -- -- -- Human L delay aging 26230157 Sen_G_0687 SEMA4C 54910 protein coding MDA-MB-231 -- Breast cancer Prevent Cell morphological analysis//SA-β-gal activity assay//Knockdown//Cell counting//Immunostaining "Cell counting assays demonstrated that knockdown of SEMA4C significantly inhibited 24 MDA-MB-231 cellular growth ability. Cell immunohistochemistry also indicated that MDA-MB-231 cells expressing shSEMA4C showed a >50% reduction in proliferation rate as assessed by anti-human PCNA staining.However, in the following days, some gradually acquired a flattened and enlarged cell shape, thus implying a pro-senescent non-apoptotic state.The senescent marker of SA-β-gal increased significantly in MDA-MB-231cells transfected with either siSEMA4C or siPLEXINB2 compared with control cells." -- -- -- -- p53-p21 -- Western blot//Knockdown "The ratio of phospho-p21Cip1–Thr145 to total p21Cip1 increased in the MDA-MB-231 cells transfected with either siSEMA4C or siPLEXINB2,suggesting that knockdown of SEMA4C or PLEXINB2 induced activation of p53, followed by activation of p21Cip1." Human HL delay aging 31308149 Sen_G_0688 ACKR3 57007 protein coding C4-2B -- Prostate cancer Prevent Cell morphological analysis//SA-β-gal activity assay//Knockdown "After one month in culture, no further proliferation could be observed, and all cells had developed a broad, flattened morphology with numerous long protrusions.In proliferation-arrested CRISPR-Cas9 mutated C4-2B colonies, strong blue staining was observed in nearly all cells indicating cellular senescence." Androgen receptor(AR) Binding CHIP//Luciferase reporter assay "Chromatin immunoprecipitation (ChIP) assays were performed on the genomic CXCR7 promoter in LNCaP cells in the presence and absence of androgen. The promoter of prostate specific antigen (PSA) was used as a positive control for AR binding and showed a significant enrichment with androgen stimulation compared to androgen deprived cells. In the CXCR7 promoter, there was a significant increase in AR binding at the predicted ARE 2 region (R1881: 2.35±0.54 vs. CDFBS: 0.49±0.09) as well as the region containing the closely spaced (7 nt separation) AREs 4 and 5 (ARE 4/5) (R1881: 1.65±0.14 vs. CDFBS: 1.11±0.12). Androgen stimulation also increased AR enrichment of the ARE 1 and ARE 3 regions, but did not reach significance. There was no change in enrichment at the ARE 6 region." EGFR -- Western blot//Histochemical staining "We observed substantially reduced CXCR7-ARRB1 and increased CXCR7-ARRB2 interactions in the CXCR7-mutant LNCaP cell line compared to WT CXCR7 LNCaP cells. These data reveal that the lost protein region containing amino acids 114 to 243 altered protein-protein interactions necessary for the observed ARRB-EGFR-CXCR7 regulatory axis, providing a potential explanation for disrupted EGF-mediated mitogenic signaling." Human HL delay aging 28596572 Sen_G_0689 TRMT9B 57604 protein coding Htrm9L Tumour sample Colorectal cancer Accelerate SA-β-gal activity assay//Immunostaining "Although some inter-experiment variability was observed for the absolute levels of senescence, hTRM9Lexpressing tumour nodules were consistently higher in SA-β-Gal activity and peaked 3 days after in vivo inoculation. Close to 80% of the hTRM9L proficient cells stained positive for SA-β-Gal activity after 3 days and up to 7 days in vivo, with a maximum of 40% of the control cells staining positive for SA-β-Gal activity 5-days post inoculation.We also detected a consistent up-regulation of the cell cycle inhibitor p21." LIN9//HIF1-a Upregulation//Upregulation Western blot//Immunostaining "We validated that the LIN9 transcript and protein is up-regulated 2-fold in hTRM9 expressing cells.First, we performed a quantitative analysis of HIF1-a protein levels in SW620, SW620-LacZ and SW620-hTRM9L expressing cells grown 3 days post inoculation on the CAM. We determined that HIF1-a protein levels were 2-fold higher in hTRM9L expressing cells.We monitored HIF1-a nuclear localization under hypoxic conditions and determined that nuclear localization was occurring at similar levels (65%) in both hTRM9L and LacZ expressing cells." -- -- -- -- Human HL delay aging 23381944 Sen_G_0690 MYC 4609 protein coding HT-1080 -- Cancer Accelerate SA-β-gal activity assay The overexpression of c-Myc resulted in increased cellular senescence in transfected HT-1080 cells which was prevented by transducing the cultures with lentiviral-p30II (HA) or through the co-expression of FLAG-tagged TIGAR. -- -- -- -- -- -- -- -- Human L delay aging 29462755 Sen_G_0691 FBXO31 79791 protein coding MCF-7 -- Aging Accelerate SA-β-gal activity assay//BrdU assay "we found that ectopic expression of FBXO31 in MCF7 cells promoted growth arrest, as evidenced by reduced proliferation and decreased DNA replication (BrdU incorporation), and induced senescence, as evidenced by positive staining for senescence-associated β-gal." MDM2//p53//ATM Downregulation//Upregulation//-- Western blot//Co-IP "The immunoblot shows that ectopic expression of FBXO31 resulted in decreased levels of MDM2, which, as expected, were accompanied by increased levels of p53 and p21.The presence of MDM2 in the FBXO31 immunoprecipitate, which was increased by lactacystin addition. The reciprocal coimmunoprecipitation experiment showed the presence of myc-FBXO31 in the MDM2 immunoprecipitate.FBXO31 failed to reduce MDM2-6A levels, confirming the essential role of ATM in FBXO31-directed degradation of MDM2" -- -- -- -- Human L delay aging 26124108 Sen_G_0692 SQSTM1 8878 protein coding PaSC -- Pancreatic ductal adenocarcinoma Prevent SA-β-gal activity assay//Knockdown//Flow cytometry//qRT-PCR "qPCR analysis corroborated the upregulation of these genes including IL-8, CXCL1, CXCL2, CCL2, and CCL20. This transcriptional change suggested an inflammatory and senescent phenotype of PaSCs. Flow cytometry analysis showed that FAP expression was increased in sqstm1 shRNA-transfected PaSCs. Consistently, β-galactosidase staining demonstrated that downregulation of sqstm1 in PaSCs led to enhanced senescence." -- -- -- -- -- -- -- -- Human HL delay aging 31182922 Sen_G_0693 SAMHD1 25939 protein coding HeLa -- Aicardi–Goutières syndrome Prevent SA-β-gal activity assay//Knockdown//Flow cytometry//Western blot "Depletion of SAMHD1 in HeLa cells by RNA interference led to increased expression of p21. This was accompanied by a delay in cell cycle progression and signs of cellular senescence, as shown by increased β-galactosidase activity." -- -- -- -- -- -- -- -- Human HL delay aging 24445253 Sen_G_0694 TOP1 7150 protein coding HCT116 -- Aging Prevent SA-β-gal activity assay//Knockdown//Flow cytometry//Clonogenic assay "We observed using clonogenic assays that sn38 induced a complete inhibition of cell proliferation. Using beta-galactosidase staining, we also noticed an induction of senescence following genotoxic treatment.control cells were synchronized in the G2 phase of the cell cycle after 48-72 hr." Aurora-A -- qRT-PCR//Western blot//Knockdown "Although this was the cell cycle stage when Aurora-A expression was supposed to be maximal, results indicated that the expression of the kinase was downregulated in response to sn38, both at the protein and mRNA levels.Finally, these experiments have also been repeated in a different colorectal cell line and sn38 also downregulated Aurora-A in HT29 cells." -- -- -- -- Human L delay aging 20682043 Sen_G_0695 TERT 7015 protein coding U937 -- Atherosclerosis Prevent SA-β-gal activity assay//Knockdown//qRT-PCR "MPM isolated from first-generation TERT-deficient mice significantly upregulated senescence associated -galactosidase activity, an established biomarker of replicative senescence. Furthermore, TERT-deficient macrophages revealed increased transcript levels of several genes causally involved in replicative senescence,26 including p16,p21, and the retinoblastoma protein." -- -- -- -- NF-κB Binding CHIP "We next performed chromatin immunoprecipitation assays using primer pairs that cover the NF-KB site at 592/580 in the human TERT promoter to determine whether NF-KB subunits are recruited to the endogenous TERT promoter. These assays confirmed that LPS, oxLDL, and TNF- induce the recruitment of both NF-KB subunits to the consensus site in the human TERT promoter. Similarly, chromatin immunoprecipitation analysis in primary MPM revealed that oxLDL induces a strong recruitment of p65 and p50 to a region encompassing an NF-KB site at 250/238 in the murine TERT promoter." Human L delay aging 21106948 Sen_G_0696 SLC4A2 6522 protein coding BEC -- Primary biliary cholangitis Prevent SA-β-gal activity assay//Knockdown "Transfection of BEC with AE2-specific siRNA that reduced AE2 expression significantly increased the proportion of SA-β-gal-positive cells (p < 0.05) and this effect was significantly inhibited by NAC (p < 0.05), indicating that the increased cellular senescence is indeed caused by the reduced level of AE2 expression." -- -- -- -- -- -- -- -- Human L delay aging 27592379 Sen_G_0697 FANCD2 2177 protein coding Fancd2+/+ epithelial cell Skin Fanconi anemia Accelerate SA-β-gal activity assay//Knockdown "To test this hypothesis, we examined the level of senescence-associated (SA)-β-galactosidase staining, a measure of in vivo senescence (Lin et al., 2010), in skin extracted from the TPA-exposed animals. Consistent with the reduced proliferation observed in TPA-treated Fancd2+/+ epithelial cells, we detected a higher accumulation of SA-β-galactosidase staining in this tissue compared to Fancd2+/-epithelium." TAp63//USP1 Upregulation//-- CHIP//Western blot "Consequently, we performed chromatin immunoprecipitation (ChIP) assays to determine whether FANCD2-Ub could directly bind to the promoter region of TAp63. The promoter regions of the p63 gene have previously been analyzed (Buckley et al., 2011; Waltermann et al., 2003). We initially divided the promoter of TAp63 into six regions(A1–A6)The nuAs predicted, A549 cells expressing USP1 shRNA showed an increase in the level of FANCD2-Ub. The reduction of USP1 expression by shRNA, and the increased FANCD2-Ub expression, inhibited the colony formation of A549 cells in soft agar and inhibited the tumor xenograft growth in nude mice.The ChIP results demonstrate that FANCD2-Ub can bind to relevant cisacting regulatory sequences of the TAp63 gene, leading to enhanced TAp63 expression and enhanced cellular senescence." -- -- -- -- Human HL delay aging 23806336 Sen_G_0698 PIP4K2A 5305 protein coding BT-474 -- Breast cancer Prevent SA-β-gal activity assay//Knockdown//Immunostaining "We observed a significant increase in reActivate oxygen species (ROS), in oxygen consumption, and in β-galactosidase staining in the BT474 cells in response to knockdown of both PI5P4Kα and β consistent with ROS-induced senescence.Decrease in cell proliferation in response to knockdown of both PI5P4Kα and β." -- -- -- -- PI3K-Akt -- Western blot//Knockdown "This increase in AKT phosphorylation could be explained by an increase in PI-3,4,5-P3 levels in response to knockdown of both PI5P4Ka and b . Surprisingly, we observed a dramatic decrease in PI-3,4-P2 in the double-knockdown cells .The levels of INPP4B were virtually undetectable in BT474 cells prior to the knockdown of PI5P4Ka and b but were quite high following the knockdown, explaining the drop in PI-3,4-P2 levels." Human HL delay aging 24209622 Sen_G_0699 PIP4K2B 8396 protein coding BT-474 -- Breast cancer Prevent SA-β-gal activity assay//Knockdown//Immunostaining "We observed a significant increase in reActivate oxygen species (ROS), in oxygen consumption, and in β-galactosidase staining in the BT474 cells in response to knockdown of both PI5P4Kα and β consistent with ROS-induced senescence.Decrease in cell proliferation in response to knockdown of both PI5P4Kα and β." -- -- -- -- PI3K-Akt -- Western blot//Knockdown "This increase in AKT phosphorylation could be explained by an increase in PI-3,4,5-P3 levels in response to knockdown of both PI5P4Ka and b. Surprisingly, we observed a dramatic decrease in PI-3,4-P2 in the double-knockdown cells.The levels of INPP4B were virtually undetectable in BT474 cells prior to the knockdown of PI5P4Ka and b but were quite high following the knockdown, explaining the drop in PI-3,4-P2 levels ." Human HL delay aging 24209622 Sen_G_0700 GNMT 27232 protein coding "HuH-7-GNMT,HuH-7-GFP,HepG2" -- Hepatocellular carcinoma Accelerate SA-β-gal activity assay//Flow cytometry//MTT assay "The growth rate of HuH-7-GNMT cells was significantly slower than that of HuH-7-GFP cells. Similar results were also observed in HepG2 cells overexpressing GNMT. Cell cycle analysis showed that, 36 h after the cells entered the cell cycle, the percentages of cells in the G2/M phase for HuH-7-GNMT cells and HuH-7-GFP cells were 23.8% and 10.4%, respectively.Furthermore, SA-β-gal assay demonstrated that HuH-7-GNMT cells had asignificantly higher rate of positive staining than HuH-7-GFP cells." DEPTOR Binding FRET-AB assay//Co-IP "Immunoprecipitation of either HAtagged DEPTOR or endogenous DEPTOR coprecipitated FLAG-tagged GNMT. In addition, we detected endogenous DEPTOR in GNMT immunoprecipitants prepared from mouse liver. The results showed that the FRET efficiency between full-length GNMT and DEP domains of DEPTOR decreased significantly. In addition, a >50% decrease of the FRET efficiency was found between full-length DEPTOR and the N-terminal of GNMT." mTOR-Raptor Activation Western blot "The results showed that overexpression of GNMT led to increases of both 4E-BP phosphorylation and cell size. In addition, overexpression of DEPTOR in HuH-7-GNMT stable cells resulted in the neutralization of the effect of GNMT on 4E-BP phosphorylation." Human L delay aging 22160218 Sen_G_0701 SIRT1 23411 protein coding BM-MSC -- Osteoporosis Prevent BrdU assay//Colony formation assay//SA-β-gal activity assay//Western blot "Compared with cells from the vehicle-treated group, resveratrol-treated cells had a higher percentage of BrdU positive cells and numbers of ALP+ CFU-f colonies. Resveratrol-treated M-MSCs from WT mice had similar numbers of ALP+ CFU-f colonies as vehicle treated M-MSCs from Sirt1TG mice, and resveratrol-treated M-MSCs from Sirt1TG mice had the highest numbers of ALP+ CFU-f colonies.Consistent with these results, p16 expression levels and senescence associated β-Gal positive areas were increased in H-BM-MSCs from old people and decreased in response to resveratrol, and these changes were blocked by the Sirt1-specific knockdown virus." Bmi1 Binding Western blot//IP The results showed that Sirt1 could bind to Bmi1.Immunoprecipitation with an anti-Bmi1 antibody showed a dose-dependent upregulation of Sirt1 protein expression associated with decreased expression of acetylated lysine. Western blot data from nuclear lysates demonstrated that the dose-dependent upregulation of Sirt1 was concomitant with the upregulation of Bmi1 expression. -- -- -- -- Human HL delay aging 30690778 Sen_G_0702 NAMPT 10135 protein coding IMR-90 -- Aging Accelerate SA-β-gal activity assay//Knockdown//Immunostaining "Inhibition of NAMPT by knockdown or by treatment with the small-molecule inhibitor FK86619 suppressed RAS-induced senescence at the time of initiation. Notably, NAMPT upregulation coincided with the upregulation of genes in the second, proinflammatory, SASP wave such as IL1B, IL6 and IL8." -- -- -- -- -- -- -- -- Human HL delay aging 30778219 Sen_G_0703 KDM4A 9682 protein coding IMR-90 -- Aging Prevent SA-β-gal activity assay//Knockdown "Compared to cells transfected with siGFP, cells depleted of JMJD2A exhibited a flattened vacuolated morphology accompanied by a positive staining to the senescence-associated β-galactosidase assay(SA-β-gal).However, the knockdown of JMJD2A led to the accumulation of PML nuclear bodies,another well- characterized senescence marker." CHD5 Binding CHIP//qRT-PCR//Knockdown "Among the two putative JMJD2A-binding sites identified by ChIP-on-chip on the CHD5 promoter, only the site located at +741 was enriched by ChIP-qPCR.JMJD2A depletion also led to a concomitant increase of CHD5 mRNA and protein.We detected an increase of H3K9(me3) levels at the CHD5 promoter after depletion of JMJD2A ." p53 Downregulation SA-β-gal activity assay//Knockdown "Transfection of siRNAs targeting JMJD2A triggered senescence in p16-depleted cells but not in p53-depleted cells, suggesting that the senescence response triggered by decreased JMJD2A levels relies primarily on the p53 pathway. Ectopic expression of JMJD2A caused a significant decrease of both p53 and p21 in Ras-expressing cells." Human L delay aging 23168260 Sen_G_0704 ABCC3 8714 protein coding HEC-TR -- Aging Accelerate Cell morphological analysis//SA-β-gal activity assay//qRT-PCR "This ABCC3-induced proliferation reduction was linked to senescence induction, as cells at p2 postinfection adopted the distinctive enlarged morphology of senescent cells and displayed both increased SA-βGal activity and induction of IL8 expression." -- -- -- -- -- -- -- -- Human L delay aging 26073088 Sen_G_0705 PIK3CA 5290 protein coding MCF 10A/H -- Breast cancer Accelerate Cell morphological analysis//SA-β-gal activity assay "Unexpectedly, we found that MCF-10A/H cells exhibited a significant increase in cell size after 96-h serum-starvation, which was accompanied with flat and enlarged morphology, suggesting cellular senescence might be induced in serum-starved MCF-10A/H cells. The fraction of SA-β-gal-positive cells increased with the duration of serum-starvation and nearly 80% of cell population under-went senescence after cells were serum-depleted for 96 h .GDC0941, a pan inhibitor of class I PI3K, significantly abrogated the induction of senescence represented by decreased SA-β-gal-positive cells." MME Upregulation SA-β-gal activity assay//Knockdown//RT-PCR//Western blot "The cluster analysis showed that the mRNA level of membrane metallo-endopeptidase (MME) increased in MCF-10A/H cells compared to parental cells and the up-regulation was further enhanced when MCF-10A/H cells were serum-starved. Induction of MME was confirmed at both RNA and protein levels in serum-deprived MCF-10A/H cells. Moreover, knock-down of MME significantly blocked the induction of senescence in serum-starved MCF-10A/H cells." PI3K-Akt-mTOR -- SA-β-gal activity assay//Western blot//Knockdown//qRT-PCR "GDC0941, a pan inhibitor of class I PI3K, significantly abrogated the induction of senescence represented by decreased SA-β-gal-positive cells, which was consistent with the down-regulation of phosphorylated AKT at S473. Similar results were obtained with A66, a PI3Kα-selective inhibitor, which was consistent with the observation that knocking down of p110α alone was able to block the induction of senescence.As AKT is the major downstream effector of PI3K signaling, we treated serum-starved MCF-10A/H cells with GSK690693, an ATP-competitive inhibitor, or MK2206, an allosteric inhibitor. Both compounds prevented the induction of senescence at concentration range that inhibited AKT activity demonstrated by phosphorylated PRAS40." Human HL delay aging 30671946 Sen_G_0706 ATF1 466 protein coding Sarcoma-iPSC MEF -- Clear cell sarcoma Accelerate Cell morphological analysis//SA-β-gal activity assay//RT-PCR "EWS/ATF1-expressing sarcoma-iPSC MEFs ceased growth and changed morphology into a large and flat shape.The senescenceassociated β galactosidase (SA β-gal)-positive cell ratio was significantly higher in EWS/ATF1-expressing MEFs than in non-expressing MEFs,suggesting that EWS/ATF1 induces premature senescence in sarcoma-iPSC MEFs." -- -- -- -- -- -- -- -- Human HL delay aging 31488818 Sen_G_0707 SERTAD1 29950 protein coding MCF-7 -- Aging Prevent SA-β-gal activity assay//Knockdown "The SLP induced by doxorubicin was not observed in p34SEI-1– expressing MCF7 cells. Furthermore, the levels of various cytoplasmic marker proteins such as fibronectin and promyelocytic leukemia, which specifically decreases in senescent cells, were lower than control values in p34SEI-1–expressing cells, suggesting that p34SEI-1 inhibits doxorubicin-induced cellular senescence in MCF7, human breast cancer cells. Cells treated with p34SEI-1-siRNA were compared for SA-β-Gal activity with those treated using scrambled RNA. SA-β-Gal activity of p34SEI-1-siRNA–treated cells that were stained 4 days after doxorubicin treatment was increased as much as control cells that were stained at 6 days, suggesting that p34SEI-1 silencing sensitizes cells to doxorubicin." -- -- -- -- -- -- -- -- Human L delay aging 19903772 Sen_G_0708 NPM1 4869 protein coding MEF -- Aging Accelerate Cell morphological analysis//SA-β-gal activity assay//BrdU assay "Fibroblasts that overexpressed NPM remained viable for several days but did not incorporate bromodeoxyuridine.Morphologically the cells became flat, enlarged and positive for acidic β-galactosidase (SA-β-Gal), a marker of senescence." p53 Upregulation Cell counting//Western blot//qRT-PCR "The physical association and colocalization of NPM and p53, and its ability to induce cellular senescence in a p53-dependent manner, suggest that NPM might regulate p53 activity directly.GFP–NPM inhibited the growth of human WI38 cells and caused a marked upregulation in the amounts of p53, hdm2 and p21 proteins.Increased transcription of hdm2 and p21 was con-firmed at the mRNA level by quantitative polymerase chain reaction." -- -- -- -- Human L delay aging 12080348 Sen_G_0709 MDM2 4193 protein coding LS8817 -- Cancer Prevent SA-β-gal activity assay//Western blot "It is well known that enforced expression of MDM2 does not increase its abundance in cycling cells; however, enforced expression did prevent the PD0332991-induced reduction in MDM2. Accumulation of phospho-Rb and cyclin A were also reduced.However, it prevented the CDK4i-induced accumulation of SA-β-gal positive cells." -- -- -- -- -- -- -- -- Human L delay aging 25803170 Sen_G_0710 ID1 3397 protein coding PC-3 -- Prostate cancer Prevent CCK-8 assay//SA-β-gal activity assay//Knockdown "Cell viability was decreased by more than 20% in siRNA-Id1 cells compared with that in nonspecific siRNA control cells, more than 30% of cells in siRNAId1 showed the appearance of enlarged blue cells in contrast to the siRNA control cells (P < 0.01)." -- -- -- -- -- -- -- -- Human L delay aging 20881502 Sen_G_0711 NF1 4763 protein coding -- Mice ear Melanoma Accelerate SA-β-gal activity assay//Knockdown "Importantly, we found that deep dermal lesions derived from control Tyr::CreER T2 ; Braf CA/+ mice stained positive for senescence associated–β-galactosidase, as has been shown in human nevi and in lesions within the Braf V600E - driven mouse model described by Dhomen and colleagues . However, senescence was not observed in Tyr::CreER T2 ; Braf CA/+; Nf1 flox/flox mice. These results are consistent with our cellular studies and indicate that mutations in Nf1 prevent Braf -induced senescence of melanocytes in mice, thereby rescuing the proliferative restriction and triggering excessive proliferation." -- -- -- -- PI3K-Akt-mTOR -- Western blot "Moreover,Nf1 mutations minimized the suppressive effects of Braf mutations on this pathway.Notably, we found that the PI3K inhibitor GDC-0941 prevented melanocytic hyperplasia in Tyr::CreER T2;Braf CA/+ ; Nf1 flox/flox mice,showing that Nf1 loss was mediating its effects in melanocytes, in part, by permitting or enhancing the activation of this pathway." Human L delay aging 23171796 Sen_G_0712 ICMT 23463 protein coding Fibroblast -- Hutchinson-Gilford progeria syndrome Accelerate Knockdown//Growth curve assay "As expected,Zmpste24?/? Icmt+/+ fibroblasts proliferated slowly and senesced prematurely. In contrast, Zmpste24?/? Icmthm/hm and Zmpste24?/? Icmt△/△ cells proliferated at rates similar to those of wildtype cells." prelamin A -- DAPI "prelamin A was mainly found at the nuclear rim in Zmpste24?/?Icmt+/+hepatocytes,colocalizing with LAP2b.In contrast,prelamin A in Zmpste24?/?Icmthm/hm hepatocytes was abundant in the nucleoplasm." Akt-mTOR -- Knockdown//Western blot "We observed higher levels of phosphorylated AKT and greater mTOR activation in Zmpste24?/? Icmthm/hm cell lysates than in Zmpste24?/? Icmt+/+ lysates, evident by increased phosphorylation of its downstream targets ribosomal protein S6 and 4E-BP1. Similar results were observed in comparisons of Zmpste24+/+Icmthm/hm and Zmpste24+/+ Icmt+/+ cells. Consistent with increased rates of proliferation, Zmpste24?/? Icmthm/hm cells had lower levels of the cyclin-dependent kinase inhibitors p27KIP1 and p21CIP1 and the tumor suppressors p16INK4A and phosphorylated retinoblastoma protein (Rb)." Human L delay aging 23686339 Sen_G_0713 SIRT1 23411 protein coding HUVEC -- Atherosclerosis Prevent SA-β-gal activity assay//Knockdown "We observed that the adenovirus-mediated SIRT1 knockdown directly induced senescence in young HUVECs. Conversely, SIRT1 overexpression significantly decreased SA-β-gal activity in senescent HUVECs, suggesting that SIRT1 protects against endothelial replicative senescence." PAI-1 Downregulation Western blot//qRT-PCR//Knockdown "PAI-1 expression was dramatically enhanced after SIRT1 knockdown at both the mRNA and protein levels in young HUVECs, while SIRT1 overexpression markedly inhibited PAI-1 expression at both the mRNA and protein levels in senescent HUVECs." -- -- -- -- Human L delay aging 25040736 Sen_G_0714 SERPINE1 5054 protein coding HUVEC -- Aging Accelerate SA-β-gal activity assay "CPAI-Q123K, HPAI-RR, and the negative control PAI-L all failed to directly induce HUVEC senescence, whereas CPAI resulted in SA-β-gal-positive staining in nearly 40% of HUVECs. Moreover, treatment of senescent HUVECs with the PAI-1 inhibitor PAI-039 significantly decreased the senescent cell ratio in a dose-dependent manner.we added PAI-039 into young HUVECs infected with Ad-U6 or Ad-SIRT1 RNAi and observed that PAI-1 inhibition significantly reversed the senescence induced by SIRT1 knockdown in HUVECs. The results showed that treatment with an exogenous stable form of PAI-1 CPAI, but not CPAI-Q123K, HPAI-RR, and PAI-L, nearly completely blocked the antisenescence effect of adenovirus-mediated SIRT1 overexpression in senescent HUVECs." -- -- -- -- -- -- -- -- Human L delay aging 25040736 Sen_G_0715 CDKN2A 1029 protein coding C6 -- Aging Accelerate SA-β-gal activity assay//Colony formation assay//Cell morphological analysis "The control cells surviving G418 selection in the colony formation assays were not altered morphologically, however we noticed the pCLp16 infected cells were flattened, large, or bi-polar. These morphological changes may suggest that the cells had entered senescence. only large or bipolar C6 cells infected with pCLp16 survived selection and, in addition, were stained blue by the SA-βGal assay." -- -- -- -- -- -- -- -- Human L delay aging 11983028 Sen_G_0716 DUSP16 80824 protein coding PLC-PRF-5 -- Aging Prevent SA-β-gal activity assay "Intriguingly, the percentage of SA-β-Gal-positive cells significantly increased upon DUSP16 silencing." Rb -- Western blot//Knockdown "In PLC/PRF/5 cells, we observed that decreased levels of Cyclin-dependent kinases (CDKs) upon DUSP16 silencing led to reduced phosphorylation of Rb,as well as reduced phosphorylation of Rb upon DUSP16 silencing." p53 -- Western blot//Knockdown "Upon DUSP16 knockdown, p53 downstream effectors were dramatically up-regulated . we confirmed that p53 phosphorylation was increased upon DUSP16 knockdown." Human HL delay aging 26381291 Sen_G_0717 RAPGEF4 11069 protein coding ESC -- Decidualization of Human Endometrial Stromal Cell Prevent SA-β-gal activity assay//Western blotR//Knockdown//Cell morphological analysis "The intensity of SA-β-Gal staining was significantly increased in the EPAC2 siRNA-treated group compared with the control.When the expression of p53 and p21 was examined in these cells, knockdown of EPAC2 suppressed p53 levels and increased p21 levels.Double knock-down of EPAC2 and p21 reduced the ratio of SA-β-Gal-positive cells when compared with single knock-down of EPAC2." CRT -- Western blot//Knockdown//qRT-PCR "Consistent with the results of LC-MS/MS analysis, EPAC2 knock-down significantly suppressed CRT expression, regardless of the presence of cAMP analogs.real-time RT-PCR analysis showed that EPAC2 knock-down inhibited expression of CRT mRNA in each group, with or without cAMP analogs." -- -- -- -- Human L delay aging 24169561 Sen_G_0718 CALR 811 protein coding ESC -- Decidualization of Human Endometrial Stromal Cell Prevent SA-β-gal activity assay//Western blot//Knockdown//Cell morphological analysis "The intensity of SA-β-Gal staining was significantly increased in the CRT siRNA-treated group compared with the control.When the expression of p53 and p21 was examined in these cells, knockdown of CRT suppressed p53 levels and increased p21 levels.Double knock-down of CRT and p21 reduced the ratio of SA-β-Gal-positive cells when compared with single knockdown of CRT." -- -- -- -- -- -- -- -- Human L delay aging 24169561 Sen_G_0719 CEBPB 1051 protein coding "PB-TRE,PB-CEBPB,LNCaP-shNTV" -- Prostate cancer Accelerate SA-β-gal activity assay//Flow cytometry//qRT-PCR//Immunostaining "Cells expressing shCEBPB or shNTV both went into G1 cell cycle arrest when cultured in androgen depleted media (ADM).Compared to control PB-TRE, PB-CEBPB cells had a significantly increase in the number SA-β-gal positive cells and the level of cell granularity, as assessed by side scatter.overexpression of C/EBPβ led to a significant increase in the transcript levels of two SASP-associated genes, IL8 and IGFBP3. When cultured in ADM, LNCaP-shNTV cells display a 2-fold increase in the number of HF positive cells.the proportion of Ki67-negative cells after one week of culture in ADM was 2-fold lower in cells lacking C/EBPβ." -- -- -- -- -- -- -- -- Human HL delay aging 25772238 Sen_G_0720 JPT1 51155 protein coding "A549,HUVEC" -- Cancer Prevent CCK-8 assay//SA-β-gal activity assay//EdU assay//qRT-PCR//Knockdown "All three cell lines showed decreased proliferation rate and increased percentage of senescence-associated SA-β-Gal positive cells.Second, decreased DNA synthesis rate was observed in HN1-KD cells by the EdU incorporation assay.LMNB1, a senescence marker indicating nuclear changes of senescent cells ,showed significantly decreased expression in both HN1-KD HUVECs and A549 cells.The result showed that HN1-KD induced down-regulation of CDK1, CCNB1 and up-regulation of IL6 in both HUVECs and A549 cells." -- -- -- -- -- -- -- -- Human HL delay aging 31257225 Sen_G_0721 HNRNPA1 3178 protein coding "HUVEC,A549" -- Cancer Prevent CCK-8 assay//SA-β-gal activity assay//Knockdown//Flow cytometry//qRT-PCR//Western blot "HNRNPA1-KD led to a higher percentage of positive SA-β-Gal stained cells and slower cell growth rate in both cell types. Besides, HNRNPA1-KD HUVECs and A549 cells also showed arrested G2/M phase, decreased LMNB1 expression, increased ROS production, and altered expression of senescence-associated molecular markers (including CDK1, CDK2, CCNB1, and IL6), resembling to the effects caused by HN1-KD." HN1-L -- qRT-PCR//Knockdown "Knockdown of HNRNPA1 or HNRNPM could promote the relative expression of HN1-L compared to total expression (HN1-L and HN1-S, labelled as HN1-T), but HNRNPA1 demonstrated the maximun effect, so HNRNPA1 was choosed for further investigation. the increased L/T ratio in HNRNPA1-KD cells could be reversed with HNRNPA1 overexpression." -- -- -- -- Human HL delay aging 31257225 Sen_G_0722 MYC 4609 protein coding "hTERT-immortalized WRN?/? Fibroblast strains AG00780,hTERT-immortalized WRN?/? Fibroblast strains AG03141" -- Aging Accelerate SA-β-gal activity assay//Microarray//Knockdown "However, within 2–3 passages, ～30%–70% of the WRN–/– cells expressed senescence-associated (SA-) β-galactosidase and lost proliferative capacity. The senescent phenotype in c-myc-transduced WRN–/– cells was also confirmed at the gene expression level by microarray analysis, which demonstrated elevated expression of several genes characteristic of replicative senescence, such as the matrix proteases. In contrast, only a small percentage of the hTERT+ WRN–/– cells transduced with a control retroviral vector expressed SA-β-gal (～1%), similarly to hTERT-immortalized normal fibroblasts (hTERT+, from two independent donors) upon MYC overexpression." WRN Binding CHIP//qRT-PCR//Co-IP "Comparable results demonstrating increased WRN A and B site binding by MYC were obtained in four different cell lines expressing deregulated c-myc.In vivo binding of MYC to the WRN promoter and a modest but consistent elevation in histone H4 acetylation, as observed for other MYC-target genes (Frank et al. 2001), was also shown by duplex PCR in the B-cell line P-493-6 that expresses a Tet-Myc-repressible gene." -- -- -- -- Human HL delay aging 12842909 Sen_G_0723 WRN 7486 protein coding hTERT+ Fibroblast -- Aging Prevent SA-β-gal activity assay//Knockdown WRN-depleted cells proliferated poorly compared to empty vector or cells expressing a control RNAi . MYC overexpression failed to rescue theproliferative capacity of WRN-depleted cells and led to a significant increase in the percentage of SA-β-gal-positive cells. -- -- -- -- -- -- -- -- Human HL delay aging 12842909 Sen_G_0724 CHEK2 11200 protein coding "BJ,Fibroblast,iG4 Terc?/?Fibroblast" Colon Telomere dysfunction Accelerate Knockdown//Western blot//Immunostaining "Infection of late‐passage, primary human BJ fibroblasts with lentiviral vectors expressing a CHK2‐directed shRNA confirmed that CHK2 knockdown abrogated the induction of senescence, resulting in positive selection of CHK2‐knockdown cells and improved proliferation rates of human fibroblast cultures at late passage. Similar results were obtained for mouse ear fibroblasts. Genetic deletion of CHK2 rescued proliferation of iG4 Terc?/? fibroblasts. Immunofluorescence staining of phosphorylated CHK1 showed nuclear staining of phospho-CHK1 in intestinal basal crypts and progenitor cells of iG4 mice. Immunofluorescence on CHK1-shRNA-treated cells confirmed the staining specificity. In addition, western blot analysis of intestinal epithelium reconfirmed the activation of CHK1 in iG4 and iG4 Chk2?/? mice." -- -- -- -- -- -- -- -- Human L delay aging 20577265 Sen_G_0725 TP53 7157 protein coding HSC -- Aging Accelerate RT-PCR//SA-β-gal activity assay//Ki67 staining//DAPI staining//Cell counting "We found a time-dependent increase in TP53 mRNA level expression upon Dox removal. We also found increased expression of CDKN2A (encoding p16INK4A) and CDKN1A (encoding p21), genes important in inducing cell cycle arrest and senescence25, after Dox was withdrawn. HSCs after Dox removal showed increased SA-β-gal (senescence-associated β-galactosidase) activity with reduced cell proliferation, measured by cell number by 4′,6-diamidino-2-phenylindole (DAPI) and quantifying cells positive for Ki67 staining." -- -- -- -- -- -- -- -- Human L delay aging 30962418 Sen_G_0726 FASN 2194 protein coding HSC -- Aging Accelerate RT-PCR//SA-β-gal activity assay//Western blot//Knockdown "FASN knockdown in senescent HSCs prevented the senescent-induced cell cycle arrest, in addition to preventing the upregulation of different markers of senescence such as SA-β-gal activity, p53, p16 and p21. Notably, FASN knockdown also inhibit the increase in the expression of mRNA levels of SASP factors IL1A, IL1B and IL6 in HSCs." -- -- -- -- mTOR -- Western blot "To our surprise,mTOR activity was repressed during senescence as shown by a decrease in the phosphorylation levels of some of its downstream targets: S6, 4EBP1 and AKT. Remarkably, senescent cells treated with C75 were able to restore mTOR signalling at levels similar to control." Human L delay aging 30962418 Sen_G_0727 TRPM7 54822 protein coding "BxPC-3,PANC-1" -- Pancreatic cancer Prevent SA-β-gal activity assay//Cell morphological analysis//qRT-PCR//Knockdown "Consistent with the observations in the micrographs with phase contrast, bright field examination reveals that both BxPC-3 and PANC-1 cells with targeted knock down of TRPM7 expression exhibit morphological features indicative of a senescent phenotype. In the BxPC-3 and PANC-1 cells transfected with anti-TRPM7 siRNA but not in the controls, SA β-gal activity was detected.In TRPM7-deficient PANC-1 cells, the mRNA levels of p16INK4A and WRN were elevated whereas that of p27CDKN1B remained unaltered. Similarly, in TRPM7-deficient BxPC-3 cells, the mRNA levels of p16INK4A and WRN were elevated by 1.4-fold and 26%, respectively." WRN -- qRT-PCR "In TRPM7-deficient PANC-1 cells, the mRNA levels of p16INK4A and WRN were elevated whereas that of p27CDKN1B remained unaltered. Similarly, in TRPM7-deficient BxPC-3 cells, the mRNA levels of p16INK4A and WRN were elevated by 1.4-fold and 26%, respectively." -- -- -- -- Human L delay aging 22166235 Sen_G_0728 HMGB2 3148 protein coding IMR-90 -- Aging Prevent Knockdown//qRT-PCR//Western blot//SA-β-gal activity assay We validated that HMGB2 expression was decreased at both the mRNA and protein levels during senescence of normal primary human embryonic fibroblast IMR90 cells induced by oncogenic RAS.knockdown or knockout of HMGB2 induced senescence in IMR90 cells . C/EBP-β//SASP Binding//-- Knockdown//CHIP-seq//CHIP//qRT-PCR "C/EBP-β is a direct target gene of HMGB2 in senescent cells, and knockdown of HMGB2 decreased C/EBP-β gene expression.Loss of HMGB2 allows for spreading of heterochromatin marks and promotes the inclusion of SASP gene loci into SAHF, which in turn represses SASP gene expression." -- -- -- -- Human HL delay aging 27799366 Sen_G_0729 SMARCD1 6602 protein coding "TIG-1,Hepatocyte,Hepatocyte" -- Aging Prevent Knockdown//Western blot//SA-β-gal activity assay "Results show that the relative number of cells with increased senescence-associated βgalactosidase (SA-β-Gal) activity, p16/p21 expression, and phospho p38 (p-p38) expression is increased in SMARCD1-silenced TIG1 cells.In contrast, cellular senescence was shown to be suppressed in senescent TIG-1 cells (61 PDL) where SMARCD1 was ectopically expressed, as evidenced by a decreased number of senescence marker-positive cells in SMARCD1-overexpressing TIG-1 cells. Furthermore, SMARCD1 expression attenuated the replicative senescence-induced growth retardation.we evaluated the effect of Smarcd1-knockdown on the cellular senescence induction in mouse primary hepatocytes, indicating that Smarcd1 knockdown also induced cellular senescence in normal hepatocytes." -- -- -- -- -- -- -- -- Human L delay aging 28868154 Sen_G_0730 HSP90AA1 3320 protein coding "IMR-90,HFF" -- Lung cancer Prevent Knockdown//SA-β-gal activity assay "Regardless of the concentration, GA treatment doubled or tripled the amount of β-galactosidase-positive senescent IMR90 fibroblasts and HFFs compared to controls .As expected, HSP90α or β knockdown by isoform-specific HSP90 siRNAs accelerated cellular senescence." p14 -- Western blot GA treatment or HSP90 ablation induced an increase in p14ARF protein levels and prolonged the half-life of the p14ARF protein under cycloheximide (CHX) treatment in the human cervical cancer cell line HeLa. -- -- -- -- Human HL delay aging 27793846 Sen_G_0731 HSP90B1 7184 protein coding "IMR-90,HFF" -- Lung cancer Prevent Knockdown//SA-β-gal activity assay "Regardless of the concentration, GA treatment doubled or tripled the amount of β-galactosidase-positive senescent IMR90 fibroblasts and HFFs compared to controls.As expected, HSP90α or β knockdown by isoform-specific HSP90 siRNAs accelerated cellular senescence." p14 -- Western blot GA treatment or HSP90 ablation induced an increase in p14ARF protein levels and prolonged the half-life of the p14ARF protein under cycloheximide (CHX) treatment in the human cervical cancer cell line HeLa. -- -- -- -- Human HL delay aging 27793846 Sen_G_0732 SIRT6 51548 protein coding HUVEC -- Aging Prevent BrdU assay//SA-β-gal activity assay//Immunostaining "SIRT6-silenced cultures showed decreased rates of proliferation and a reduction in the cell fraction passing through the S-phase.SIRT6-silenced cultures also displayed an increase in the proportion of SA-β-gal+ cells.In SIRT6-depleted HUVEC, there was a significant increase in γH2AX foci." -- -- -- -- p21 -- Western blot This analysis showed higher levels of p21 expression in SIRT6-depleted cells compared with controls. Human L delay aging 23201774 Sen_G_0733 MIF 4282 protein coding MSC -- Myocardial infarction Prevent Knockdown//Western blot//SA-β-gal activity assay "MIF-siRNA treatment of young MSCs significantly downregulated MIF expression, while it significantly elevated p53 and p21 expression. Furthermore, MIF-siRNA treatment remarkably increased the number of SA-β-gal-positive cells among young MSCs.While the overexpression of MIF in aged MSCs enhanced MIF expression, it reduced p53 and p21 expression. Moreover, the percentage of SA-β-gal-positive cells was greatly reduced in MIF-aged MSCs compared with aged MSCs." Beclin1//LC3-I/II//p62 Upregulation//Upregulation//Downregulation Western blot "Autophagy has recently been found to inhibit cellular senescence.Overexpression of MIF in aged MSCs significantly induced autophagy, as manifested by the elevated expression of Beclin1 and LC3I/II and the reduced expression of p62." -- -- -- -- Human L delay aging 31881006 Sen_G_0734 ZBTB48 3104 protein coding pMSC -- Aging Accelerate Western blot//SA-β-gal activity assay//qRT-PCR "Overexpression of TZAP in P2 pMSCs resulted in an increase in the percentage of SA-β-gal-positive cells by 17% (from 6 to 23%). Western blot analysis showed that compared to the control vector, TZAP over-expression in pMSCs significantly increased the protein levels of P21 and P16. Consistent with this finding, qRT-PCR also revealed upregulated transcript levels of p21 and p16Ink4a." -- -- -- -- p53 Upregulation Western blot "After overexpression of TZAP in pMSCs, western blot analysis showed that overexpression of TZAP led to increased levels of ARF, P53 and P21 but decreased levels of MDM2 compared to those in pMSCs transduced with the control vector. In contrast, knockout of TZAP in pMSCs decreased ARF, P53, and P21 levels but increased MDM2 levels ." Human HL delay aging 30845965 Sen_G_0735 NFATC1 4772 protein coding prostate epithelial cell -- Prostate cancer Prevent Knockdown//SA-β-gal activity assay//Immunostaining "There was a marked decrease in the expression of the senescence marker p21 in PCre/+;RT/+;TN/+;Ptenfl/fl samples when compared with the PCre/+;Ptenfl/fl mice. p21 staining was predominantly nuclear in PCre/+;Ptenfl/fl prostates (63.6 ± 7.95%). In contrast, nuclear p21 expression was absent in PCre/+;RT/+;TN/+;Ptenfl/fl (4.2 ± 1.30%) prostates, where cytoplasmic p21 was occasionally observed.To further confirm that NFATc1 activation overcomes PTEN lossinduced cellular senescence, we stained for senescence-associated β-galactosidase (SA-β-gal) activity in the prostates. Control and PCre/+;RT/+;TN/+ prostates showed very few senescent cells, 1% and 6.66 ± 0.5%, respectively. In contrast, 65.6 ± 8.7% cells within the PCre/+;Ptenfl/fl prostates were SA-β-gal+. Such SA-β-gal+ cells in the PCre/+;RT/+;TN/+;Ptenfl/fl prostates were markedly reduced to 5.8 ± 1.3%." -- -- -- -- PTEN-AKT Activation Immunostaining "Interestingly, all double mutants (PCre/+;RT/+;TN/+;Ptenfl/fl) with both PTEN deficiency and NFATc1 activation developed significantly larger tumors in all prostate lobes when compared with mice of the same age with either Pten deficiency or NFATc1 activation alone . The average prostate weight in double mutants (6026.24±1946.85?mg) was increased 17.41-fold when compared with the controls (346.85±36.66?mg), 15.45-fold when compared with mice with NFAT activation alone (390.28±73.16?mg), 7.35-fold when compared with Pten null mice. Histopathological analyses revealed that Pten null mice and mice with NFATc1 activation alone had PIN at this time, whereas double mutants already had poorly differentiated prostatic adenocarcinoma. Although levels of pAKT were low in prostates from controls and mice with only NFATc1 activation, increased expression of pAKT was apparent in PCre/+;Ptenfl/fl and PCre/+;RT/+;TN/+;Ptenfl/fl samples, indicating that the PI3K-AKT pathway was activated in prostates with PTEN loss." Human HL delay aging 26477312 Sen_G_0736 MAD2L1 4085 protein coding MKN45 -- Gastric cancer Prevent SA-β-gal activity assay//qRT-PCR "With this experiment, we determined that the fraction of SA-β-gal-positive cells increases 3d after Mad2 depletion. When we analyzed its activation after 72 h of PTX treatment, we observed an increase in p53 mRNA levels in cells lacking Mad2.IL-6 (and not IL-8) expression was attenuated when Mad2 was decreased." -- -- -- -- -- -- -- -- Human L delay aging 25483095 Sen_G_0737 BUB1B 701 protein coding MKN45 -- Gastric cancer Prevent SA-β-gal activity assay//qPCR "With this experiment, we determined that the fraction of SA-β-gal-positive cells increases 3d after BubR1 depletion. When we analyzed its activation after 72 h of PTX treatment, we observed an increase in p53 mRNA levels in cells lacking BubR1. Both IL-6 and IL-8 mRNA levels were increased during senescence in the absence of BUB1B." -- -- -- -- -- -- -- -- Human L delay aging 25483095 Sen_G_0738 NUPR1 26471 protein coding "A549,H460" -- Lung cancer Prevent Knockdown//Western blot//SA-β-gal activity assay//Flow cytometry//BrdU assay//Colony formation assay "In this regard, NUPR1 depletion in A549 or H460 cells caused a marked increase in the number of GLB1 (galactosidase beta 1)-positive cells. Consistent with the induction of GLB1, NUPR1 depletion induced G0/G1 cell cycle arrest, with significant upregulation of the key cell cycle inhibitors CDKN1A/p21Cip1 and CDKN1B/ p27Kip1, but not CDKN2A/p16INK4a. NUPR1 knockdown also inhibited cell growth, as evidenced by BrdU incorporation and colony formation assays." -- -- -- -- -- -- -- -- Human HL delay aging 29130426 Sen_G_0739 HRAS 3265 protein coding IMR-90 -- Cancer Accelerate Western blot//SA-β-gal activity assay Cells infected with the retrovirus expressing HRASG12V fail to proliferate and stained positive for senescence-associated β-galactosidase (SA-βgal) activity. -- -- -- -- -- -- -- -- Human HL delay aging 24618719 Sen_G_0740 CUX1 1523 protein coding IMR-90 -- Cancer Prevent Western blot//SA-β-gal activity assay Co-expression of p200 CUX1 enabled RAS expressing cells to proliferate normally and prevented SA-βgal activity. OGG1 Activation Western blot "The enzymatic activity of OGG1 was greatly stimulated by recombinant CUX1 proteins containing one or more Cut repeat domain(s): CR2CR3HD, CR3HD, and CR1CR2." -- -- -- -- Human HL delay aging 24618719 Sen_G_0741 ASXL1 171023 protein coding MEF -- Aging Prevent SA-β-gal activity assay//DAPI staining//SAHF//Cell morphological analysis//Western blot//Microarray//qRT-PCR " SA-β-gal staining was significantly greater in two different passages of Asxl1-null MEFs than in WT MEFs. Consistently, more SAHF were formed in Asxl1-null MEFs as determined by DAPI staining.More senescence was induced at passage 6 of Asxl1-null MEFs because of growth retardation in the later stages. In addition, Asxl1-deleted MEFs displayed an enlarged and flattened shape (data not shown).Notably,the up-regulation of another Cdk inhibitor, p16Ink4a (a hallmark of cellular senescence),was observed in Asxl1-null cells, whereas p21Waf1 was down-regulated at the protein and RNA levels.In addition to p16Ink4a, other senescence -associated genes such as p57Kip2, Mmp1, and Pai1 were also up-regulated as shown by microarray analysis and RT-qPCR." E2F//EZH2 --//-- qRT-PCR//Co-IP "Most of the E2F target genes were significantly down-regulated in Asxl1-null.To explore the link between ASXL1 and EZH2,we first measured the physical interaction by co-IP analysis. As reported previously, Flag-EZH2 interacts with endogenous ASXL1." Akt-E2F -- Western blot//CHIP "A significant decrease in p27Kip1 phosphorylation was observed in Asxl1-null MEFs, which was likely due to Akt inactivation. As shown by IP assays using HEK293 cells treated with IGF-1, we demonstrated that p27Kip1 interacts with both ASXL1 and AKT1 regardless of IGF-1–induced AKT phosphorylation. Consistent with reports shown above, IGF-1 treatment induced the cytoplasmic export of p27Kip1 in normal MEFs, whereas the IGF-1 effect was abolished in Asxl1-null MEFs; p27Kip1 was thus retained in the nucleus. Consequently, we observed a gradual down-regulation of Rb phosphorylation, but no change in the Rb level, during culture of Asxl1-null MEFs. In response to IGF-1, no effect of Asxl1 was observed on E2F1 binding to the Ccna2 promoter, while the IGF-1–repressed Rb binding was recovered in Asxl1-null MEFs after Rb activation." Human HL delay aging 28701722 Sen_G_0742 RELA 5970 protein coding Human low-grade PanIN cell Pancreata Pancreatic ductal adenocarcinoma Accelerate Western blot//SA-β-gal activity assay//qPT-PCR "Quantification of SA-β-Gal–positive cells in low-grade mPanIN lesions from Kras and Kras RelA mice demonstrated loss of SA-β-Gal activity in Kras RelA mPanIN.In addition, the senescence marker decoy receptor 2 (DCR2; also known as TNFRSF10D) was significantly lower in Kras RelA pancreata than in Kras pancreata on both an mRNA expression and a protein level.GSEA demonstrated a loss of the SASP signature in Kras RelA mice." CXCL1 -- Western blot//SA-β-gal activity assay "Of the 40 cytokines and chemokines represented in this panel, CXCL1 (also known as KC) was most robustly downregulated in PDEC Kras treated with JSH-23. The decrease in CXCL1 protein levels was corroborated by an increased SA-β-Gal activity in PDEC Kras incubated with CXCL1 for 48 hours .In Kras pancreata, Cxcl1 mRNA expression was moderately to strongly present in the cytoplasm of most duct cells in mPanIN lesions, in acinar cells around mPanIN lesions, and in immune cells.Cxcl1 mRNA was present in immune cells, only faintly present in a few duct cells, and absent in the majority of acinar cells in Kras RelA pancreata." -- -- -- -- Human HL delay aging 27454298 Sen_G_0743 NTN4 59277 protein coding "U251MG,pcDNA3" -- Glioblastoma Prevent SA-β-gal activity assay "We observed significantly fewer senescent NTN4-overexpressing cells compared with mock control cells.Thus, NTN4 overexpression delayed U251MG cell senescence induced by H2O2." -- -- -- -- -- -- -- -- Human HL delay aging 30514230 Sen_G_0744 EGF 1950 protein coding U251MG -- Glioblastoma Prevent SA-β-gal activity assay "On the 4th day after H2O2 treatment, we observed less senescence in U251MG cells treated with EGF compared with non-EGF treated cells. On the 7th day after H2O2 treatment, the number of senescent cells in the EGF treatment group was significantly less than that in the non-treatment control group.There was a significant difference in U251MG senescence between cells treated and not treated with EGF for 7?days. With EGF (40?ng/ml) treatment, senescence of U251MG cells induced by H2O2 decreased. The concentration of 40?ng/ml EGF inhibited U251MG cell senescence efficiently." NTN4 Upregulation qRT-PCR//Immunostaining We observed that NTN4 expression were significantly increased upon EGF stimulation at both mRNA level and protein level. -- -- -- -- Human HL delay aging 30514230 Sen_G_0745 COPS5 10987 protein coding MEF -- Cancer Prevent SA-β-gal activity assay//Cell morphological analysis//Western blot "Under the microscope, 4OHT‐treated cells appeared flatter, a typical feature of senescent cells. We, therefore, assayed for senescence‐associated (SA) β‐galactosidase (Gal) activity, another marker of premature senescence.Lower panels show that 4OHT‐treated CSN5f/‐p53?/?Ras+CRE‐ER MEFs were positive for SA‐β‐Gal activity.As cells underwent senescence, the expression of the CDK inhibitors p21, p27, and p16 was upregulated and hypo‐phosphorylated Rb protein was accumulated, whereas the level of Skp2 was maintained." -- -- -- -- PI3K-Akt -- Western blot//Knockdown "Although the total expression levels of ERK1, ERK2, and Akt were maintained, their activating phosphorylation was modulated by knockout of the CSN5 gene. The Activate form of ERK1 and 2 was reduced, whereas the activating phosphorylation of Akt (serine 473 and threonine 308) was increased after treatment with 4OHT. Furthermore, the phosphorylation of certain substrates of Akt was enhanced in cells deprived of CSN5.We observed the same phenotype when we used a chemical inhibitor more specific to PI3 kinase, LY294002." Human L delay aging 23127558 Sen_G_0746 CNOT6 57472 protein coding MCF-7 -- Aging Prevent SA-β-gal activity assay//Knockdown "Consistent with the role of IGFBP5, knockdown of Ccr4a/Ccr4b caused a significant increase in senescence-associated β-galactosidase staining as compared with control or Caf1a/Caf1b knockdown." IGFBP5 -- Knockdown//qRT-PCR "We confirmed enhanced expression of IGFBP5 (approximately threefold), CLEC3A (approximately threefold), SEMA3E (approximately twofold), MAPK10 (approximately twofold), CDH18 (approximately twofold), and LMO3 (approximately eightfold) upon Ccr4a/Ccr4b knockdown." p53 -- Western blot//Knockdown "Interestingly, while the overall levels of both total p53 as well as p53 acetylated at Lys-120 were significantly increased in Ccr4a/Ccr4b knockdown cells, the fraction of p53 acetylated at Lys-120 was not increased." Human HL delay aging 21233283 Sen_G_0747 CNOT6L 246175 protein coding MCF-7 -- Aging Prevent SA-β-gal activity assay//Knockdown "Consistent with the role of IGFBP5, knockdown of Ccr4a/Ccr4b caused a significant increase in senescence-associated β-galactosidase staining as compared with control or Caf1a/Caf1b knockdown." IGFBP5 -- Knockdown//qRT-PCR "We confirmed enhanced expression of IGFBP5 (approximately threefold), CLEC3A (approximately threefold), SEMA3E (approximately twofold), MAPK10 (approximately twofold), CDH18 (approximately twofold), and LMO3 (approximately eightfold) upon Ccr4a/Ccr4b knockdown." p53 -- Western blot//Knockdown "Interestingly, while the overall levels of both total p53 as well as p53 acetylated at Lys-120 were significantly increased in Ccr4a/Ccr4b knockdown cells, the fraction of p53 acetylated at Lys-120 was not increased." Human HL delay aging 21233283 Sen_G_0748 RBM38 55544 protein coding MEF -- Lymphoma Prevent SA-β-gal activity assay//Knockdown We showed that the number of cells stained positive with senescence-associated β-galactosidase (SA-β-gal) was markedly increased by lack of RNPC1 regardless of DNA damage . Quantitative analysis indicated that SA-β-gal-positive cells were increased by threefold to eightfold upon loss of RNPC1 (47.5% in RNPC1?/? MEFs vs. 5.5% in RNPC1+/+ MEFs and 6.5% in RNPC1+/? MEFs in the absence of doxorubicin; 64.5% in RNPC1?/? MEFs vs. 18% in RNPC1+/+ MEFs and 19.5% in RNPC1+/? MEFs in the presence of doxorubicin). -- -- -- -- p53 Downregulation Western blot "We showed that p53 was highly induced by RNPC1 deficiency, especially upon treatment with doxorubicin, consistent with the observation. Similarly, transient expression of RNPC1a, but not RNPC1b, inhibited p53 expression in a dose-dependent manner. Furthermore, we showed that upon treatment with doxorubicin or nutlin-3, ectopic expression of RNPC1a markedly attenuated p53 accumulation in MCF7 and RKO cells ." Human L delay aging 21764855 Sen_G_0749 PYGL 5836 protein coding U87 -- Cancer Prevent SA-β-gal activity assay//Cell morphological analysis//Flow cytometry//Immunostaining//Knockdown "Analysis of the cell-cycle profile after PYGL knockdown in U87 cells revealed a higher proportion of cells in G1 phase with concomitant decreases in both S phase and G2/M phase, as compared to control cells.Following PYGL depletion, U87 cells underwent characteristic morphological changes (i.e., enlargement and flattening) that were indicative of cellular senescence.Following PYGL knockdown, we observed an increase in both DEC1 and SA β-gal staining." -- -- -- -- p53 -- Knockdown//Western blot//Cell counting Knockdown of p53 prevented the decreased growth rate phenotype normally associated with PYGL depletion. Human L delay aging 23177934 Sen_G_0750 ENO1 2023 protein coding CFPAC-1 -- Pancreatic cancer Prevent SA-β-gal activity assay//Cell morphological analysis//Western blot//Flow cytometry "The cell-cycle profile analysis after 24 h serum deprivation revealed a significant increase in the number of ENO1-silenced cells in G2/M phase, a concomitant decrease of cells in G1 phase and no difference in the number of cells in S phase .there was a decrease in expression of the negative regulator of the cyclin D/CDK complex p18 (INK4C) after ENO1 silencing. ENO1-silenced cells showed characteristic morphological changes, such as enlargement and flattening, which were indicative of cellular senescence, confirmed by β-galactosidase staining ." -- -- -- -- -- -- -- -- Human L delay aging 26734996 Sen_G_0751 NCOA4 8031 protein coding MEF -- Aging Prevent SA-β-gal activity assay//Western blot//Knockdown//Immunostaining "NCOA4?/? cells showed increased activation of the apical DDR kinases ATM and ATR, accumulation of nuclear foci stained for pS/TQ ATM/ATR substrates, and increased phosphorylation of p53 on S15. Furthermore, NCOA4?/? cells displayed an increased number of γ-H2AX- and 53BP1-positive cells compared to NCOA4+/+ MEFs.Consistently, NCOA4?/? MEFs displayed reduced PDL (population doubling level) accumulation compared to both NCOA4+/+ and NCOA4+/? cells, rapidly exhausted replicative potential, and after four passages, entered premature senescence, as shown by increased β-galactosidase staining (p < 0.001)." -- -- -- -- -- -- -- -- Human L delay aging 24910095 Sen_G_0752 KLF6 1316 protein coding "LN-229,BTSC233" -- Glioblastoma Accelerate SA-β-gal activity assay//Flow cytometry "Prolonged expression of KLF6-wt, but not KLF6-sv1, induced a senescent-like phenotype in both LN229 and BTSC23 cells, highlighted by β-galactosidase staining. Cell cycle analysis revealed accumulation of cells in phase G1–G0 and concomitant reduction of cells in phases S and G2/M, upon KLF6-wt expression.In contrast, KLF6-sv1 overexpression prolonged S-phase. In BTSC23 cells, KLF6-wt overexpression led to accumulation of cells in G2/M .Consistent with the G1 arrest observed in LN229 cells, KLF6-wt overexpression led to upregulation of CDKN1A expression in LN229 and BTSC23 cells ." -- -- -- -- NF-κB Downregulation Western blot "Downregulation of NF-κB targets MMP9, OLIG2 and YKL40 was confirmed by western blot." Human HL delay aging 28166199 Sen_G_0753 ABI3BP 25890 protein coding GBC -- Gallbladder cancer Accelerate SA-β-gal activity assay//EdU assay//Transwell assay//Western blot "The results demonstrated that compared with cells manipulated with vector-NC, cells manipulated with ABI3BP-vector displayed markedly enhanced ability of cell viability, weakened abilities of migration and invasion and promoted SA-β-gal activity, as well as significantly down-regulated expression of Ki67 and PCNA." -- -- -- -- -- -- -- -- Human HL delay aging 31174563 Sen_G_0754 ATM 472 protein coding "DLD1,HCT116" -- Colorectal cancer Prevent SA-β-gal activity assay//Knockdown//Flow cytometry "Intriguingly, cell cycle analysis showed that ATM deletion induced a significant G2/M arrest of DLD1 and HCT116 cells under normal conditions.Moreover, we observed a significantly higher number of senescent ATM-/- cells compared with senescent ATM+/+ cells, as determined by SA-β-gal staining ." B56γ2 Binding Western blot//Co-IP "Indeed,western blot analysis showed that B56γ2 expression was significantly increased in ATM-/-cancer cells.Intriguingly, the coimmunoprecipitation experiments showed that ATM and B56γ2 co-immunoprecipitated reciprocally." Chk1-p53-p21 -- Western blot//Knockdown "The results showed that in ATM-/- cells, Cdc2 phosphorylation on Tyr15, which is indicative of decreased Cdc2 activity, was significantly increased . In addition, the phosphorylation of the upstream regulator Chk1 was significantly increased in ATM-/- cells, whereas the phosphorylation of Chk2 was not altered . In accordance with this phenomenon, the phosphorylation levels of p53 and p21, two key regulators of senescence [23], were significantly increased in ATM-/-cells, indicating that ATM deficiency promotes p53/p21-induced senescence." Human HL delay aging 28093285 Sen_G_0755 ASAH1 427 protein coding A375 -- Melanoma Prevent SA-β-gal activity assay//Cell morphological analysis//Flow cytometry//PI staining "Only 2% of ASAH1-null cells entered the G2 phase, compared to 25% scramble-treated control cells. The remaining ASAH1-null cells were found in G1 (63%) or S (35%) phase. Similarly, ASAH1 deletion was accompanied by the appearance of a phenotype characterized by senescence-like cell morphology and accumulation of senescence-associated β-galactosidase." -- -- -- -- -- -- -- -- Human L delay aging 28785021 Sen_G_0756 RHEB 6009 protein coding Articular Chondrocyte Bone Osteoarthritis Prevent SA-β-gal activity assay//DAPI staining "RHEB- 9 overexpression not only recovered the morphology of the DCs, but also reduced senescence by 10 35–40 %. RHEB-overexpression also decreased ROS levels." COL2A1//SOX9 Upregulation//Upregulation Western blot "Importantly, 11 RHEB -overexpression increased the level of COL2A1, which was almost negligible in non-transfected DCs. SOX9 expression also increased with RHEB -overexpressing DCs." -- -- -- -- Human L delay aging 31229684 Sen_G_0757 AGER 177 protein coding -- Lung Aging Prevent SA-β-gal activity assay//Knockdown//Immunofluorescence//Histological staining "The absence of RAGE was associated with an accumulation of fibrotic tissue (as evidenced by Masson's trichrome staining) and senescent lesions, as evidenced by β-Gal-staining . The accumulation of these senescent lesions was also observed in other tissues of RAGE?/? mice.When markers of senescence associated cellular properties were studied, an increase of IL-6 by ～50% and an almost 3-fold increase of γH2AX and 53BP1 was seen, while pATM increased by ～50%. This is consistent with DNA damage associated senescence and a senescence associated pro-inflammatory phenotype in cells and lungs of RAGE?/? mice , indicating an on-going persistent DNA damage signaling." -- -- -- -- -- -- -- -- Human L delay aging 28977635 Sen_G_0758 SIRT1 23411 protein coding ECFC -- Preterm Prevent Western blot//SA-β-gal activity assay//BrdU assay//Cell morphological analysis "Twenty-four hours after transient transfection, SIRT1 overexpression was associated with a significant decrease in the level of the senescence-associated p16INK4a protein;Consistently,transfection of PT-ECFCs with the SIRT1 vector significantly reduced SA-β-gal activity and senescence associated morphological changes, compared with PT-ECFCs transfected with an empty vector;SIRT1 overexpression accelerated PT-ECFC proliferation." -- -- -- -- -- -- -- -- Human L delay aging 24518759 Sen_G_0759 FGF21 26291 protein coding BVSMC -- Cerebrovascular aging Prevent SA-β-gal activity assay//Western blot Treatment of recombinant FGF21 (100 nM) significantly attenuated the positive area of SA-β-gal staining;FGF21 treatment decreased the NBS1. p21//TRF2 Downregulation//Upregulation Western blot "FGF21 also inhibited the induced p21 expression by AngII.Siah-1 is an E3 ubiquitin ligase degrading TRF2，These results suggest that FGF21 enhances TRF2 expression via inhibiting Siah-1 signaling in hBVSMCs.Interestingly, FGF21 partly but significantly inhibited the upregulation of ROS and superoxide anion induced by Ang II ." p53//Siah-1//AMPK Downregulation//Downregulation//Activation Western blot//SA-β-gal activity assay FGF21 supplement substantially depressed the both the total p53 and phospho-p53 levels induced by AngII ；FGF21 inhibited Siah-1 expression induced by AngII.FGF21 markedly increased phosphorylation of AMPK in hBVSMCs.SA-β-gal staining assay showed that blockade of AMPK activation by specific inhibitor of AMPK Compound C almost totally abolished the anti-aging action of FGF21. Human L delay aging 27364911 Sen_G_0760 SIRT1 23411 protein coding Clara Lung Chronic obstructive pulmonary disease Prevent SA-β-gal activity assay//Western blot "We found that the level of SIRT1 was decreased, whereas senescence-associated β-gal activ-ity and p21 expression were increased in lungs of COPD patients compared with nonsmokers.CS exposure significantly increased the levels of prosenescent proteins (i.e., p21, p16, and p53) and SA–β-gal activity in lungs of Sirt1+/– mice versus WT littermates, whereas these levels were lowered by Sirt1 overexpression." FOXO3//p21 --//-- SA-β-gal activity assay//Western blot "Foxo3–/– mice exhibited heightened levels of p21, p16, p27, and SA–β-gal activity in lungs in response to CS exposure.However, there was no significant change in SA–β-gal activity in lungs of p21–/– mice in response to CS exposure, or along with sirtinol treatment.Moreover, FOXO3 acetylation was increased in Sirt1+/– mice,but lowered in Sirt1 Tg mice exposed to CS for 6 months." -- -- -- -- Human L delay aging 22546858 Sen_G_0761 IHH 3549 protein coding B-MSC -- Aging Prevent SA-β-gal activity assay//RT-PCR//Western blot//Cell morphological analysis//Colony formation assay//PI staining//Flow cytometry "Surprisingly, the count of BMSC cells stained by SA-β-gal stain increased in cells treated with IHH siRNA.Consistently, aging-related genes, p16, p53, SA-β-gal, and mTOR were downregulated after treatment with rIHH.The IHH siRNA transfected BMSC showed more transparency, slight enlargement,and decreased in cell count compared to the negative control.IHH siRNA-transfected BMSC failed to form colonies contrary to the negative control.G0/G1 cell cycle arrest was associated with BMSC of IHH siRNA." -- -- -- -- ROS-mTOR-4EBP1 Downregulation//-- Western blot//Flow cytometry//Colony formation assay//Knockdown "We observed down-regulation of P53 and P16 associated with inhibition of mTORand ROS pathways.The cell cycle results showed that inhibition of mTOR and ROS pathways restricted the G0/G1 cell cycle arrest caused by IHH silencing .The colony forming ability of BMSC caused by IHH knockdown was improved after inhibition of mTOR and ROS in the presence of siRNA IHH;As we expected, knockdown of IHH induced 4EBP1 and p70S6K1/2 phosphorylation but rIHH protein treatment down-regulate the phosphorylation process.Our findings presented anti-aging activity for IHH in BMSC through down-regulation of ROS/mTOR pathways." Human L delay aging 32235006 Sen_G_0762 NAMPT 10135 protein coding Adipose stromal cell Adipose Aging Prevent Lifespan assay We started injecting EVs purified from young-to-middle age (4–12-month-old) mice once a week into female mice at 26 months of age. supplementation with EVs purified from young-to-middle-aged mice significantly extended the lifespan of aged mice. -- -- -- -- -- -- -- -- Human L delay aging 31204283 Sen_G_0763 CALCA 796 protein coding EPC Blood Aging Prevent SA-β-gal activity assay//Telomerase activity assay CGRPI lentivirus transduction significantly reduced SA-β-gal positive senescent cells and elevated the activity of telomerase in AngII-treated EPCs. Klotho Upregulation Western blot//qRT-PCR "Interestingly, the levels of both Klotho mRNA and secreted Klotho protein were remarkably increased by CGRPI over-expression." -- -- -- -- Human HL delay aging 20832068 Sen_G_0764 ATG7 10533 protein coding keratinocyte Skin Aging Prevent Microarray PQ treatment induced a transcriptional signature of strong cell cycle arrest and DNA damage signaling in the Atg7 deficient cells. p53//p21//CDK1//H2AX --//--//--//-- Western blot//qRT-PCR//Immunostaining "p53 and the downstream mediator p21 were induced by PQ on mRNA and protein level, and the induction was increased in the knockouts on protein level for both proteins.Using qPCR we could verify that the knockout cells showed higher baseline expression of Cdk1.Using WB we could show that this was reflected on protein level, with a stronger Cdk1 signal in untreated KO. We exposed the cells to UVA, which did not cause a significant rise in positive nuclei in WT cells, but did so in KO cells." -- -- -- -- Human HL delay aging 28012437 Sen_G_0765 FOXQ1 94234 protein coding HUCMSC Umbilical cord Alzheimer's disease Prevent CCK-8 assay//Cell morphological analysis//SA-β-gal activity assay//qRT-PCR//Western blot//PI staining "Cell viability was significantly enhanced in the P15-FOXQ1 group compared with that in the P15-vector group on day 4.Meanwhile, the morphology of cells in the P15-FOXQ1 group was changed slightly compared with those in the P3 group,with the spindle shape maintained in most cells. The number of SA-β-gal-positive cells was noticeably reduced in the P15-FOXQ1 group.Compared with the P15-vector group,expression of positive senescence-associated genes such as p16,p21 and p53 was down-regulated at the mRNA level. At the mRNA and protein level,Expression of negative senescence-associated genes, such as SIRT1 and PCNA, was up-regulated.A significantly higher number of cells in the S phase and M phase was detected in the P15-FOXQ1 group compared with that in the P15-vector group." -- -- -- -- -- -- -- -- Human L delay aging 29500491 Sen_G_0766 KNDC1 85442 protein coding HUVEC Umbilical cord Aging Accelerate SA-β-gal activity assay The number of positive SA-β-Gal staining observed in the HUVECs following the transfection of the KNDC1?adenovirus vector significantly increased when compared with that in the control group. -- -- -- -- p53 Upregulation Western blot "However, a significant increase in the expression of p-p53 was observed in HUVECs that overexpressed KNDC1 when compared with the NT-adenovirus-transfected control cells." Human L delay aging 29568929 Sen_G_0767 FOXO3 2309 protein coding HUC-F2 -- Aging Prevent SA-β-gal activity assay//qRT-PCR Proliferative potential and senescence-associated β-galactosidase (SA-β-Gal) activity were then monitored. Our results clearly showed that the dominant Activate form of FOXO3a (FOXO3aTM) significantly promoted proliferation and inhibited the onset of replicative senescence in HUC-F2 cells in a manner similar to SIRT1 [6].We detected a significant elongation of telomere length in HUC-F2 cells transduced with FOXO3aTM. c-Myc//hTERT Upregulation//Upregulation Luciferase reporter assay//qRT-PCR "We found that FOXO3a increased the promoter activity of c-MYC and the transcription of the c-MYC gene. These results suggest that FOXO3a activates c-MYC expression, which, in turn,results in enhanced quantities of c-MYC at the hTERT promoter,enhanced transcriptional activation of c-MYC and, as a consequence, upregulation of hTERT gene expression." -- -- -- -- Human L delay aging 25000517 Sen_G_0768 CCN1 3491 protein coding -- Skin Aging Accelerate Atomic force microscopy imaging "This increase of CCN1 was associated with constitutively reduced type I collagen, and constitutively elevated MMP-1 in forearm chronically sun-exposed prematurely aged human skin." IL-1β Upregulation qRT-PCR//ELISA "Elevated expression of CCN1 in dermal fibroblasts resulted in significant upregulation of IL-1βmRNA and protein levels.Importantly, knockdown of CCN1 induction significantly reduced UV irradiation induction of IL-1β mRNA and protein levels, indicating that induction of IL-1β is dependent, in part, on induction of CCN1." -- -- -- -- Human L delay aging 23881607 Sen_G_0769 TRIM28 10155 protein coding IMR-90 -- Aging Accelerate Crystal violet assay//Immunostaining//BrdU assay//SAHF//Knockdown "We observed that TRIM28 depletion resulted in increased cell growth upon OIS induction,similar to what was observed upon p53 knockdown.TRIM28 knockdown also resulted in less cells presenting features characteristic of senescence such as senescence-associated heterochromatic foci.A higher percentage of cells with depleted TRIM28 levels incorporated BrdU 6 days upon 4OHT induction, suggesting that depletion of TRIM28 partially prevented the effects of OIS." p16 -- Immunostaining//knockdown p16INK4a levels were lower upon knockdown of TRIM28. -- -- -- -- Human L delay aging 25160591 Sen_G_0770 ARG2 384 protein coding HUVEC -- Aging Accelerate SA-β-gal activity assay//Western blot "The senescence markers such as the number of SA-β-gal positive cells,the levels of p53-S15 and p21Cip1 as well as levels of VCAM1 and ICAM1 are significantly augmented by Arg-II overexpression." eNOS -- Western blot//Immunostaining "Conversely, in nonsenescent cells, adenovirus-mediated ectopic expression of a wild-type (WT) Arg-II cDNA as verified by immunoblotting,leads to eNOS-uncoupling, i.e., impaired NO production (DAF-2DA staining) and enhanced intracellular O2-generation (DHE staining),which is significantly inhibited by the eNOS inhibitor L-NAME." -- -- -- -- Human L delay aging 22928666 Sen_G_0771 RPS6KB1 6198 protein coding HUVEC -- Aging Accelerate SA-β-gal activity assay//Western blot "S6K1 increases the number of positively stained SA-β-gal cells, elevates p53-S15 and p21Cip1 levels, and enhances VCAM1 and ICAM1 expression, demonstrating that persistent hyperActivate S6K1 promotes endothelial senescence and inflammation." Arg-II Upregulation qRT-PCR "Indeed, in young endothelial cells, overexpression of a constitutively Activate S6K1 mutant (HA-S6K1ca), but not the inActivate S6K1 mutant, which is confirmed by immunoblotting with the anti-S6K1 antibody that detects both mutants, enhances Arg-II mRNA and protein levels paralleled with increased arginase activity." -- -- -- -- Human L delay aging 22928666 Sen_G_0772 JUND 3727 protein coding Aortic endothelial cell Aorta Aging Prevent Knockdown//qRT-PCR//SA-β-gal activity assay//Western blot//Immunostaining "Telomerase activity was blunted in young JunD?/? compared with age-matched WT mouse aorta.This finding indicated a vascular senescence phenotype in young animals lacking JunD .β-Galactosidase staining further supported the early occurrence of vascular aging in JunD?/? mice. Accordingly, the expression of aging markers such as tumor suppressor p53 and the cyclin-dependent kinase inhibitor p16INK4a was increased in these mice." p47phox//NOX2//NOX4 --//--//-- Immunostaining//Western blot "In contrast,the NADPH oxidase subunits p47phox, Nox2, and Nox4 were already upregulated in young JunD?/? mice and further increased with aging." -- -- -- -- Human L delay aging 23410942 Sen_G_0773 IGF1 3479 protein coding "MCF-7,IMR-90" -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis//BrdU assay "IGF-1 treatment of IMR90 or MEF cells led to appearance of cells with feature characteristic of premature cellular senescence, including an enlarged flat cell morphology and increased β-Galactosidase (SA-β-Gal) activity.By contrast, cells treated with IGF-1 remained morphologically large and flat, with marginal BrdU incorporation ." -- -- -- -- SIRT1-p53 Activation Western blot "IGF-1 significantly inhibited SIRT1 deacetylase activity.We confirmed comparable SIRT1 protein input levels by western blot analysis.IGF-1 treatment led to a marked increase in p53 protein levels in serum-starved MCF7, U2-OS, and IMR90 cells.IGF-1 treatment led to a substantial increase in p53 protein half-life, compared with the control." Human L delay aging 25070626 Sen_G_0774 SIRT6 51548 protein coding HCA2 -- Aging Prevent Immunostaining SIRT6 reduced the number of γH2AX foci. PARP1 -- Western blot//Autoradiography "Instead, we used the specific PARP1 inhibitor PJ34. Importantly, PJ34 had no inhibitory effect on either deacetylation or mono-ADP ribosylation activities of SIRT6.In the presence of PJ34, SIRT6 overexpression had no effect on HR,indicating that SIRT6-mediated rescue of HR in aging cells is dependent on PARP1." -- -- -- -- Human L delay aging 22753495 Sen_G_0775 SIRT1 23411 protein coding "B-MSC,A-MSC" Bone and Adipose Aging Prevent Knockdown//BrdU assay//PI staining//SA-β-gal activity assay "Compared to control, the knockdown of SIRT1 markedly slowed down the growth rate of both types of MSC, as shown by plotting a curve of the cell growth and by the use of a BrdU incorporation assay.In B-MSCs, SIRT1 knockdown significantly decreases the percentage of cells in S phase,while A-MSCs have significantly more cells in the G0/G1 phase and fewer cells in the S phase and G2–M phase after SIRT1 knockdown.However, in cells from later passages, more SA-β-gal+cells were observed in those cultures where SIRT1 had been knocked down." p16 -- Western blot "However, in late culture passages, p16 but not p21 is accumulated faster in B-MSCs where SIRT1 has been knocked down.Conversely, overexpression of SIRT1, but not its dominantnegative mutant H363Y , efficiently delayed the accumulation of p16." -- -- -- -- Human L delay aging 22038097 Sen_G_0776 PTEN 5728 protein coding -- Human islet Aging Accelerate Knockdown//Immunostaining The percentage of Ki67 positive cells was dramatically increased in the Pten null mice that are 3 months old and older. -- -- -- -- PI3K-Akt//cyclin D1-E2F-Ezh2-p16 Downregulation//-- Western blot "A primary biochemical function of PTEN is to inhibit the action of PI3K.We show that phosphorylation of the PI3K effector, serine/threonine kinase AKT, is dramatically induced in the Pten null islets compared with the Con ones, whereas the amount of p-ERK is minimally altered.Elevated cyclin D1 leads to the activation of E2F through phosphorylation of retinoblastoma (RB) proteins. We found that overexpression of E2F1 led to downregulation of p16ink4a expression, whereas knockdown of E2F1 robustly induced expression of p16ink4a.We found that expression of E2Fs1-4,especially E2Fs1-3, resulted in the dramatic induction of an Ezh2 promoter activity." Human L delay aging 23826727 Sen_G_0777 RECQL4 9401 protein coding Fibroblast Mouse tail Rothmund–Thomson syndrome Prevent SA-β-gal activity assay//Immunostaining "Recql4HD fibroblasts expressed more SA-β-gal than wild-type fibroblasts.In addition, the average number of 53BP1 foci was approximately threefold higher under 3%oxygen and fourfold higher under 20% oxygen in cells from old Recql4HDcells compared with cells from old wild-type cells. The average number of γH2A.X foci per cell was two- or threefold more in Recql4HDcells than in wild-type cells." -- -- -- -- -- -- -- -- Human HL delay aging 24832598 Sen_G_0778 E2F1 1869 protein coding MEF Embryo Aging Accelerate BrdU assay//SA-β-gal activity assay//Knockdown "The ability of MEFs to proliferate decreases with cell passage; knocking out E2F1 attenuates this effect. The proliferation of passage 5 WT MEFs was greatly reduced compared with E2F1 KO MEFs, with all cells visualized by Hoechst stained DNA but DNA synthesis was greatly reduced only in WT cells.by passage 5 a significantly greater proportion of WT MEFs were testing positive for senescence compared with E2F1 KO MEFs, indicated by SA-β-gal activity." FOXO3 Downregulation Luciferase reporter assay//Immunostaining "The reporters alone show some activation of the FKRE-luciferase gene, presumably from endogenous FOXO1/3 proteins. This activation is significantly repressed when the E2F1 plasmid is co-transfected.Moreover, the levels of intracellular ROS,as measured by dichlorofluorescein (DCF) levels, whether in the steady state or under oxidative stress, were significantly reduced in E2F1 KO MEFs compared with WT cells." -- -- -- -- Human L delay aging 25344604 Sen_G_0779 E2 1489080 protein coding HeLa -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis "The parental HeLa cells infected with the E2 virus expressed the expected senescent phenotype including growth arrest, increased cell size and flattening, elevated autofluorescence and high level SA-β-gal activity." -- -- -- -- -- -- -- -- Human HL delay aging 16626397 Sen_G_0780 IGF1 3479 protein coding Myocyte -- Cardiomyopathy and heart failure Prevent Western blot//Telomere length assay "IGF-1 interfered with the age-dependent increases in myocyte size, telomeric shortening and p16INK4aand p53 proteins." -- -- -- -- PI3K-Akt Activation Western blot "Akt protein (total) remained constant and did not differ in WT and TG myocytes. However, phospho-Akt levels decreased in WT and increased in TG myocytes from 4 to 20 to 22 months." Human L delay aging 14726476 Sen_G_0781 UBE2I 7329 protein coding U2OS -- Aging Prevent Growth curve assay Under senescence promoting conditions there is a clear delay for the onset ofsenescence in U2OS when cells express the wild type form of UBC9 while the K49R variant shows no effect and undergoes senescence like the control cells. PML Binding Growth curve assay "As expected, both Ubc9-PML and Ubc9K49R-PML fusion proteins localized to nuclear bodies.Expressing either Ubc9-PML or Ubc9K49R-PML fusion proteins counteracted the senescence induced by PML as compared to GFP-PML fusion in control cells ." -- -- -- -- Human L delay aging 29773808 Sen_G_0782 HGF 3082 protein coding "hE-MSC,BM-MSC" -- Liver disease Prevent qRT-PCR//FISH "RTL was analyzed after treating hE-MSCs with a neutralizing HGF antibody.Telomere length was decreased to 50% and PDT was delayed to 80 hr upon loss of HGF function in hE-MSCs.Interphase telomere fluorescence in situ hybridization (FISH) showed that treatment of hBM-MSCs with rHGF increased the number and intensity of PNA foci in the nucleus.After normalization to AIB1, a reference gene with only a single copy on the chromosome, the relative mtDNA copy number was 1.5-fold higher in rHGF-treated cells than in control cells." RAD51 Upregulation HR assay//Western blot "When we treated hBM-MSCs with rHGF, the activity and protein level of RAD51 increased.When we blocked HGF in hE-MSCs using a neutralizing antibody,the protein level of RAD51 decreased." -- -- -- -- Human L delay aging 29398486 Sen_G_0783 MCL1 4170 protein coding HCT116 -- Colorectal cancer Prevent Immunostaining "Impressively, DPI (similar to NAC) caused a robust abrogation of ROS generation in Mcl-1 deficient cells as compared with the Mcl-1 proficient cells during CIS conditions after 24 hours of culture with doxorubicin, a time point that significant differences in ROS production can be observed." NOX4 Downregulation Western blot//Co-IP Immunoblot analysis using NOX4 specific antibody shows that NOX4 is predominantly up regulated in the mitochondrial fraction in CIS-sensitive cells under doxorubicin treatment in the absence of Mcl-1.Mcl-1 has a unique ability to inhibit ROS production by preventing the up regulation of the pro-oxidant NOX4. -- -- -- -- Human L delay aging 28423654 Sen_G_0784 MAPK1 5594 protein coding Fibroblast -- Prostate cancer Accelerate SA-β-gal activity assay//Knockdown//Western blot "ERK2 knockdown in cells expressing RasV12 inhibited the induction of senescence-associated β-galactosidase (SA-β-Gal), PML bodies, and DNA damage foci.Oncogenic ras engaged the p53/p21, p16INK4a/RB, and p38MAPK pathways in primary cells, and this was efficiently prevented by knockdown of ERK2.The induction of several senescence-associated cytokine genes by RasV12 was also efficiently blocked by ERK2 knockdown." -- -- -- -- -- -- -- -- Human HL delay aging 23599344 Sen_G_0785 SOD2 6648 protein coding Chondrocyte Bone Cartilage degeneration Prevent Safranin O/Fast Green staining//Flow cytometry Histological analyses by the OARSI score revealed that Sod2 cKO joints exhibited a significant loss of safranin-O staining in all layers of both articular cartilages in Sod2 cKO mice.The control joint showed the loss of safranin-O staining without morphological changes in the superficial layer of MFC and MTP .Sod2 cKO chondrocytes demonstrated significantly increased superoxide generation via flow cytometry with DHE and MitoSOX stainings. -- -- -- -- -- -- -- -- Human L delay aging 26108578 Sen_G_0786 LMNA 4000 protein coding -- Embryo Aging Prevent SA-β-gal activity assay "The zLMNA-MO2 morphants showed a high intensity of SA-β-gal at 6 dpf, whereas zLMNA-MO1 morphants did not show significantly detectable SA-β-gal activity." -- -- -- -- -- -- -- -- Human L delay aging 21479207 Sen_G_0787 SOD3 6649 protein coding Fibroblast Skin Aging Prevent Survival curve SOD3R213G Tg mice exhibited a shortened life span with their hair turning gray upon aging. -- -- -- -- -- -- -- -- Human HL delay aging 25927599 Sen_G_0788 DKC1 1736 protein coding MEF Embryo X-linked dyskeratosis congenita Prevent SA-β-gal activity assay//Flow cytometry//Lifespan assay "In 3% oxygen, △15 cells grew more slowly and entered senescence earlier than WT cells.△15 cells accumulated more ROS than WT cells and the difference became very significant with more PDs, even when cell .There was a significantly higher number of foci in D15 cells, and the difference was greater in high oxygen." -- -- -- -- -- -- -- -- Human L delay aging 21241452 Sen_G_0789 IRS2 8660 protein coding Astrocyte Brain Huntington disease Accelerate Survival curve "The body weights of R6/2?Irs2ntg mice increased normally until 7 weeks, but afterward declined rapidly, and half the mice died between 11 and 12 weeks of age. Based on Cox regression,neuronal Irs2 in R6/2?Irs2ntg mice significantly increased the risk of death 3.6-fold compared with R6/2 mice;sex of the mice was not a significant covariate.The fore limb grip of R6/2 and R6/2?Irs2ntg mice was significantly weaker than those of control and R6/2?Irs2+/–?Irs2βtg mice." FOXO1 -- Western blot "The fractionation of brain homogenates and immunoblotting confirmed that phosphorylated FoxO1 was largely cytoplasmic in R6/2 and R6/2?Irs2ntg mice, whereas FoxO1 was weakly phosphorylated and most strongly detected in the nuclear (laminin-containing) fractions from R6/2?Irs2+/–?Irs2βtg brains." -- -- -- -- Human L delay aging 21926467 Sen_G_0790 TP53 7157 protein coding MEF -- Tumor Accelerate SA-β-gal activity assay//Cell morphological analysis "The shorter survival of nontumor-bearing p53S18Amice suggests that their aging process could be accelerated due to the Ser18mutation. In fact, these mice exhibited heightened expression of several factors associated with aging, such as inability to heal, premature graying, chronic alopecia, and lordokyphosis. The number of positive blue cells increased with each passage of p53S18A cells, compared with wild-type cells. By passage 3,30% of p53S18A cells stained positive for β-gal, whereas none of the wild-type cells stained positive . Also, the cells seemed to be mostly large flat cells, which is consistent with cells having exited the cell cycle." -- -- -- -- -- -- -- -- Human L delay aging 18089799 Sen_G_0791 ING1 3621 protein coding Hs68 -- Aging Accelerate DAPI staining//Flow cytometry//SA-β-gal activity assay "The occurrence of SAHF in senescent cell nuclei (panel B) and highlights the similarity of nuclear phenotype and DNA staining patterns between senescent cells and cells expressing high levels of INGla.Examining the effect of ING1a on cell cycle distribution showed that overexpression of INGla induced cell cycle arrest increased progressively with time despite culturing in complete medium and the percentage of primary Hs68 cells in the G1 phase.Expression of ING1a efficiently induced the expression of SA-β-gal activity.In contrast, cells overexpressing INGla acquired a senescent-like flattened phenotype and a DNA staining pattern similar to that seen in SAHF." -- -- -- -- p16-pRb Activation Western blot ING1a expression substantially increased both pl6 and pRb levels. Human L apoptosis 18691180 Sen_G_0792 PTEN 5728 protein coding BM-MSC -- Systemic Lupus Erythematosus Accelerate SA-β-gal activity assay//Flow cytometry//Knockdown "There were less SA-β-gal-positive cells when PTEN expression was knocked down in BM-MSCs from SLE patients, but it has less effect in the control group’s BM-MSCs. The further quantitative analysis indicated the number of SLE patients’ BM-MSCs increased in si-PTEN-transfected group compared to the untreated group from the third day .Cell-cycle analysis revealed that G1 phase arrest was observably reversed in si-PTEN-transfected SLE BM-MSCs." -- -- -- -- p27 Upregulation Western blot//Immunofluorescence "In the BM-MSCs from SLE patients, we found that the expression of PTEN was up-regulated, and the phosphorylation of Akt was reduced. Meanwhile, the higher expression of PTEN and the lower expression of p-Akt in SLE BM-MSCs were confirmed by immunofluorescence. The expression of cell-cycle regulator p27kip1 was determined. The results showed that p27kip1 increased markedly in BM-MSCs from SLE patients and nuclear fluorescence intensity was enhanced. The expression of p27kip1 decreased significantly in SLE BM-MSCs transfected with si-PTEN ." Human L apoptosis 25649549 Sen_G_0793 BTK 695 protein coding "EJp53,HDF" -- Aging Accelerate Growth curve assay//SA-β-gal activity assay "Inhibition of BTK by chemical or genetic approaches increased cell proliferation in EJp53 induced to senesce. Indeed, the percentage of cells positive for senescence associated (SA)-β-gal, a widely used marker of senescence (32), was significantly lower when BTK was inhibited, and less cells showed the morphological changes typical of senescence." -- -- -- -- p53 Activation Western blot//RT-PCR "We found that BTK protein levels were elevated after inducing p53 expression in these cells. Moreover, the colon cancer cell line HCT116, which has wild-type p53, also showed a p53-dependent BTK induction after being treated with DNA damaging agents (the oxidant tBH and doxorubicin), both at protein and mRNA levels. We transfected BTK into EJp53 and observed that it elevated the levels of p53 protein induced by tet removal." Human L apoptosis 27630139 Sen_G_0794 KRT24 192666 protein coding Primary human epidermal keratinocyte Skin Aging Accelerate MTS assay//EdU Assay//Flow cytometry//SA-β-gal activity assay "The results indicated that K24 overexpressed keratinocytes showed decreased proliferation rate, as compared with control cells. Furthermore, the actual proliferative capacity of the K24 overexpressed keratinocytes was then determined by EdU assay. The percentage of proliferating cells from the K24 overexpressed cells was 43% after 72 h of incubation, whereas 78% being detected for the control cells.The result showed that the transfection of K24 decreased the DNA synthesis and induced a G1/S growth phase arrest, as evidenced by 84.09% in the experimental group, and 63.94% in the vector control cells. SA-β-galactosidase staining showed a 3-folds more positive blue cells in K24 overexpressed keratinocytes than those in control cells ." -- -- -- -- -- -- -- -- Human L apoptosis 28362807 Sen_G_0795 DLK1 8788 protein coding WI-38 -- Aging Prevent SA-β-gal activity assay//Flow cytometry//Knockdown "Cells infected with the Sh-hDLK2 lentiviral construct exhibited a significantly slower rate of proliferation when compared to control cells,suggesting that depletion of DLK inhibited cell proliferation.Our results showed that loss of DLK results in a diminution of cells in S phase, an increased proportion (～20%) of cells in G1 phase and an unchanged percent-age of cells in the G2/M phase.More than 70% of cells infected with the lentivirus expressing a human DLK shRNA (Sh-hDLK2) showed elevated SA-β-Gal activity, whereas control cells failed to exhibit β-Gal staining." -- -- -- -- ERK//p53-p21 --//-- Western blot//Knockdown "In DLK-depleted cells, we noted a substantial decrease in ERK phosphorylation level relative to control , indicating that ERK activity in WI-38 cells is dependent on DLK. we observed that p53 and p21 expression was significantly up-regulated in DLK-depleted cells as compared to cells infected with the control lentiviruses." Human L apoptosis 21893036 Sen_G_0796 SAE2 816686 protein coding "HCT116,U2OS" -- Cancer Prevent Knockdown//SA-β-gal activity assay//Flow cytometry//PI staining//BrdU assay "SAE2 and UBC9 shRNAs (sh1 and sh2) potently blocked colony formation in vitro.By PI staining and flow cytometry, we observed increased sub-G1 cell population in HCT116 cells upon conditional SAE2 knockdown , suggesting some cells are undergoing apoptosis.By BrdU incorporation assay, we observed a decreased percentage of cells in S phase in SAE2 knockdown U2OS cells.SAE2 knockdown HCT116 cells also showed a multi-nucleated phenotype with enlarged and flattened morphology which is commonly observed in senescent cells. This cell population stained positive for SA-β-Gal activity." -- -- -- -- SUMO Activation SA-β-gal activity assay//Flow cytometry//DAPI staining//Western blot "As further confirmation that the non-silencible SAE2 can functionally rescue the SUMO pathway activity, we performed the same sub-G1 apoptosis assay and SA-β-Gal staining. The wildtype SAE2, but not the C->A enzyme dead SAE2 mutant, partially rescued shSAE2 induced multinucleation and senescence,and associated cell death.SUMO substrate and SUMOylation is important for TopoIIα activity in vitro [35–37]. Western blots in HCT116 cells expressing control or SAE2 shRNA revealed endogenously SUMOylated TopoIIα species, and knockdown of SAE2 inhibited TopoIIα SUMOylation." Human L apoptosis 25860128 Sen_G_0797 IFNG 3458 protein coding Melanocyte -- Vitiligo Accelerate Flow cytometry//SA-β-gal activity assay "IFN-γ significantly decreased the cell viability in a dose dependent manner. Cell cycle analysis results demonstrated that IFN-c at both concentrations caused the accumulation of melanocytes at G1 phase, while decreased the percentage of cells at S and G2/M phases.In contrast, melanocytes after persistent IFN-γ treatment became large, flat in shape with shorter and fewer dendrites, and some cells were highly pigmented.We also noticed a significantincrease of SA-β-gal stain ing, a marker of senescence, in melanocytes with 7 days of IFN-γ stimulation." p21/WAF1 Upregulation Western blot//SA-β-gal activity assay "Immunoblotting analysis indicated that protein level of p21 was greatly elevated with the increasing duration of IFN-γ treatment,while p16 level didn’t change during the experiment.P21 siRNA treatment suppressed the IFN-γ-induced increase of SA-β-gal staining." JAK2-STAT1 -- MTS assay//Western blot//SA-β-gal activity assay//Flow cytometry "In the presence of IFN-γ, the cell viability of control siRNA transfected melanocytes was significantly inhibited. Significantly, JAK2 or STAT1 siRNA efficiently restored the viability of melanocytes. Immunoblotting results confirmed that JAK2 or STAT1 siRNA, but not JAK1 siRNA inhibited the increase of p21 induced by IFN-. Moreover, IFN-γ-induced SA-β-gal staining increase in melanocytes was blocked by JAK2 and STAT1 siRNAs.IFN-γ treatment caused obvious elevation of intracellular ROS, and the effect of IFN-γ was dose-dependent manner." Human L apoptosis 24681574 Sen_G_0798 CTSK 1513 protein coding "MEF,NHDF" -- Aging Prevent SA-β-gal activity assay//Cell morphological analysis//BrdU assay NHDF lacking cathepsin X exhibited a flattened cell body and a significant increase in size in comparison to control cells expressing normal amounts of cathepsin X. Quantification of SA-β-gal in NHDF lacking cathepsin X clearly demonstrated a significant increase both in the number of stained cells and in the intensity of staining .Cathepsin X-deficient MEF displayed a reduced cell growth when assayed by two different cell proliferation assays employing different DNA labeling techniques. The number of BrdU(+) cells which had gone through cell division (2 N DNA content) was significantly lower 24h after the pulse in cells lacking cathepsin X indicating a delayed G1 entry. -- -- -- -- -- -- -- -- Human L apoptosis 21616554 Sen_G_0799 MDM2 4193 protein coding H460 -- Lung cancer Prevent SA-β-gal activity assay//Flow cytometry "Missense oligonucleotide alone caused apoptosis in 5.6 ± 0.6% (mean F SD) cells and radiation treatment increased this to 16 ± 1.3%. The anti-MDM2 oligonucleotide alone had a greater effect than the missense oligonucleotide with 18 ± 0.2% apoptotic cells.Missense oligonucleotide caused 2.7±0.2% and 7.0 ± 0.6% (mean ± SD) of senescent cells at 0 and 5 Gy, whereas anti-MDM2 –treated cells showed significantly greater senescence with 5.3 ± 0.4% and 42.7 ± 1.5% (P < 0.001 missense compared with anti-MDM2 oligonucleotide)." p53//p21 Upregulation//Upregulation Western blot "H460 cells pretreated with missense ASODN followed by 5 Gy showed an increase in MDM2,p53, and p21 expression at 24 hours, compared with H460 treated with missense ASODN alone. " -- -- -- -- Human L apoptosis 16093429 Sen_G_0800 CDKN1C 1028 protein coding "HepG2,SNU398 HCC" -- Hepatocellular carcinoma Accelerate SA-β-gal activity assay//Cell morphological analysis//Flow cytometry//Western blot "Induction of P57 reduced proliferation of HepG2 and SNU398 HCC cells, as demonstrated by growth curve analysis.FACS analysis revealed that P57 transfected cells accumulate in G1 phase of cell cycle. Seventy-two hours post-P57 transfection, cells began to change the morphology, becoming enlarged and flattened, and they adopted a senescent phenotype when compared to vector-only cells .In accordance with the senescence phenotype, induction of P57 in HepG2 and SNU398 led to the identification SA-β-gal positive cells.An increase in P16 protein levels was also observed in HepG2 cells." -- -- -- -- Hes1 -- RT-PCR//Western blot "Notch3-and Notch1-silenced cells showed a down-regulation of Hes1 target gene together with an up regulation of P57 mRNA and protein levels .Down-regulation of Hes1 significantly increased P57 mRNA and protein levels in both analyzed cell lines .In HepG2 cells, but not in HepG2, with siRNA to Hes1, DNA of the P57 promoter region could be specifically detected in the Hes1-immunoprecipitated DNA complex from formaldehyde-treated cells, indicating Hes1 occupancy at the P57 promoter in vivo." Human L apoptosis 22705236 Sen_G_0801 SPHK2 56848 protein coding -- "Spleen,Skin,Testes tissue" Lung cancer Prevent SA-β-gal activity assay//Knockdown "SphK2-/-mice exhibited accelerated senescence and aging phenotype measured by increased β-gal expression in their paws (37) at generation 6, and decreased weight of testes atgeneration4 or 5 compared with WT and SphK1-/-mice (generation matched controls). Examination of spleen,skin, and testes tissues at generation 6 using H&E staining also confirmed increased senescence and aging phenotype in SphK2-/-compared with WT mice." -- -- -- -- p16 -- Immunofluorescence//Western blot "ShRNA-mediated knockdown of SPHK2 induced caspase-3, and ectopic expression of p16 attenuated this process compared with Scr-shRNA– expressing A549 cells." Human HL apoptosis 29748384 Sen_G_0802 GKN1 56287 protein coding "AGS,MKN1,AGSGKN1,MKN1GKN1" -- Gastric cancer Accelerate SA-β-gal activity assay//qPCR "Average telomere length and telomerase activity decreased significantly in GKN1-transfected AGS and MKN1 cells, compared to those in mock-transfected cells. Furthermore, unlike mock-transfected cells, AGSGKN1 and MKN1GKN1 cells that stably expressed GKN1 demonstrated shortened telomeres and diminished telomerase activity with additional passages.These stable transfectants from passage 5 showed significant shortening of telomeres, as well as decreased telomerase activity and hTERT mRNA expression in a time-dependent manner. The mean percentage of SA-β-gal-positive AGSGKN1 and MKN1GKN1 stable cells increased by 13% and 5.5% at 24 hr and by 24% and 19% 48 hr (P = 0.0134 and P = 0.0247), respectively." c-Myc//hTERT//TRF1 Downregulation//Downregulation//Upregulation qPCR//Western blot "GKN1 directly bound to the c-myc protein in the immunoprecipitation assay and down-regulated its expression, as well as inhibited its binding activity to TRF1.Interestingly, c-myc mRNA expression was positively correlated with hTERT expression, whereas GKN1 expression was inversely correlated with c-myc and hTERT mRNA expression in 35 gastric cancer tissues. GKN1 protein expression was positively and inversely correlated with TRF1 and c-myc protein expression." -- -- -- -- Human L apoptosis 25344918 Sen_G_0803 PAPSS2 9060 protein coding MCF-7 Tumor tissue Breast cancer Prevent Cell morphological analysis//Colony formation assay//Flow cytometry//SA-β-gal activity assay "MCF7 human breast cancer cells treated with PAPSS2 Si were fewer in number and showed poor colony-forming ability compared with control siRNA (Con Si)-transfected cells. PAPSS2-depleted cells accumulated in the G1 phase with no increase in the proportion of cells either in the subG1 phase or that were positive for propidium iodide staining. PAPSS2-depleted cells displayed hallmarks of senescent cells such as enlarged and flattened morphology and SA-β-Gal staining.As PAPSS2 was depleted, the cells showed a loss of phosphorylated pRb with changes of cell cycle regulatory protein levels and accumulation of p53 and p21." -- -- -- -- FGFR-AKT-p53-p21 -- SA-β-gal activity assay//Western blot "PAPSS2-mediated premature senescence, including a loss of phosphorylated pRb, a decrease in cell number, and an increase in SA-β-Gal positivity, was blocked in both p53?/?and p21?/?HCT116 human colon cancer cells.We confirmed that P APSS2 depletion induced augmented FGFR/AKT activation and p53/p21 accumulation in both A549 and HDF cells . The overexpression of a kinase-inActivate (KI) mutant of FGFR1 (FGFR1-KI) attenuated AKT phosphorylation and p53/p21 accumulation in PAPSS2-depleted cells." Human HL apoptosis 26250908 Sen_G_0804 PTEN 5728 protein coding "LN-18,U87,U251,U373,LN428" -- Glioma Prevent Cell morphological analysis//SA-β-gal activity assay//Western blot "We first examined relative cell numbers and cellular morphologies, and observed that all cell types had decreased cell numbers in a dose-dependent manner following IR .Furthermore, microscopic analyses indicated that U87, U251, and U373 cells were positive for senescence-associated β-galactosidase (SA-β-Gal), a hall-mark of senescence, and for senescent morphology (large flattened shape).Cyclin-dependent kinase inhibitor p21, one of senescence markers, was dramatically increased in PTEN-deficient U87, U251, and U373 cells, but not in PTEN-proficient LN18 and LN428 cells, and cleaved poly (ADP-ribose) polymerase (PARP), which is an indicator of the biochemical changes due to caspase activation during apoptosis was detected only in LN18 and LN428 cells ." -- -- -- -- Akt-ROS-p53-p21 -- Western blot//SA-β-gal activity assay//Flow cytometry "U87 cells were characterized by gradual increases in phospho-AKT, wt p53, and p21. AKT depletion also shifted cells from premature senescence to apoptosis as a response to IR , as observed in wt PTEN-expressing cells. AKT depletion resulted in decreases in both SA-β-Gal activity and intracellular ROS levels.In p53siRNA transfected cells before IR exposure, p21 expression was not induced , and cells showed decreased SA-β-Gal activity. Cells transfected with p21 siRNA before IR exposure showed phenotypes similar to those transfected with p53 siRNA." Human L apoptosis 21072054 Sen_G_0805 UBTD1 80019 protein coding "HDF,MKN45" Tumor tissue Gastric cancer Accelerate qRT-PCR //Western blot//Flow cytometry//SA-β-gal activity assay//Trypan blue staining//Knockdown "UBTD1 knockdown increased cell proliferation and down-regulated p53 protein. We further analyzed cell cycle and apoptosis by flow cytometry and dead cells by Trypan blue staining, and found that UBTD1 overexpression indeed induces G1-stage arrest, apoptosis and an increase in dead cells. Frozen tumor tissue slices were stained for SA-β-gal marker and a higher proportion of senescent cells were present in the UBTD1 overexpression group (p<0.05)." -- -- -- -- Mdm2-p53 Downregulation Western blot//qRT-PCR//SA-β-gal activity assay "We treated MKN45 cells with nutlin-3, a known MDM2 antagonist that stabilizes p53, and found that nutlin-3 upregulated p53 and UBTD1 in a dose-dependent manner.We found that exogenous UBTD1 can pull down both endogenous Mdm2 and p53 , suggesting that these three proteins can form a complex and that UBTD1 might regulate p53 via Mdm2. We overexpressed Mdm2 in UBTD1-overexpressing cells, and found that Mdm2 overexpression could reverse UBTD1-mediated up-regulation of p53 expression ." Human HL apoptosis 25382750 Sen_G_0806 RBX1 9978 protein coding "U87,H1299" -- Cancer Prevent Cell morphological analysis//SA-β-gal activity assay//Flow cytometry "Morphologic observation revealed that LT-ROC–infected U87 cells were in size with flattened shape. Indeed,～25% of LT-ROC–infected cells, but <2% of the control cells, were positively stained with SA-β-gal.Our FACS analysis also revealed that among the remaining cell populations, not undergoing apoptosis, ～50% to 60% of LT-ROC1–infected U87 cells were arrested in the G2-M phase of the cell cycle, compared with ～15% to 20% of control cells at 120 hours postinfection." -- -- -- -- -- -- -- -- Human L apoptosis 19509229 Sen_G_0807 CYP24A1 1591 protein coding Primary renal proximal tubule epithelial cell Kidney Diabetes Accelerate SA-β-gal activity assay//Flow cytometry "CYP24A1 overexpression induced senescence (increased G1 arrest) in normal conditions and induced apoptosis (increased sub-G1 fraction) in high glucose conditions. We did in fact see increased cellular senescence in cells overexpressing CYP24A1 that exceeded cellular senescence induced by high glucose alone, as shown by increased numbers of blue-stained cells ." -- -- -- -- -- -- -- -- Human L apoptosis 23119081 Sen_G_0808 TERT 7015 protein coding MSC -- Aging Prevent SA-β-gal activity assay//Flow cytometry//TUNEL assay//CCK-8 assay "Compared with the control group, the downregulation of hTERT induced cellular senescence, decreased cell proliferation and the percentage of cells in the S phase and increased the percentage of apoptotic MSCs." -- -- -- -- PI3K-Akt Upregulation Western blot "We found that the downregulation of hTERT significantly decreased p-PI3K and p-AKT expression in the MSCs, whereas the upregulation of hTERT significantly increased p-PI3K and p-AKT expression in the MSCs; no changes were observed in the levels of total PI3K and AKT in either group.The MSCs were treated with LY294002 (Sigma-Aldrich), a PI3K inhibitor, and the hTERT expression levels were then measured by western blot analysis. The results revealed that LY294002 significantly inhibited hTERT expression." Human HL apoptosis 26178664 Sen_G_0809 BMP7 655 protein coding MCF-7 -- Breast cancer Accelerate Cell morphological analysis//Western blot//Telomerase activity assay "In the BMP7 treated cell cultures, we observed the cells characteristics of enlarged and flattened cell morphology, greater cytoplasm/nuclear ratio, and expressions of cell senescence markers such as β-galactosidase and p16 (Janzen et al., 2006;Molofsky et al., 2006).Treatment of MCF-7 cells with BMP7 (30 ng/mL, 15 h in every two-day for two weeks) resulted in a marked increase in the incidence of cell senescence. The increase in cell senescence in the BMP7-treated cultures was associated with reduced cell numbers and protein concentrations (not shown), decreased telomerase activity. The inhibition of telomerase activity in these cells was by 60%–70%.Consistent with cell senescence, BMP7-treated cell cultures showed increased p53, p21 and p16." hTERT Downregulation Flow cytometry The extent of BMP7-induced hTERT gene promoter inhibition was similar to that induced by BMP7 in MCF-7 cells.Overexpression of BMP RII receptor similarly blocked BMP7-induced hTERT gene repression in PMC42 breast cancer cells. Smad3 -- Immunofluorescence//Luciferase reporter assay "Incubation of cultured MCF-7 cells with BMP7 for different periods of time from 10 min to 2 h resulted in Smad3 phospho rylation. Predominant cytoplasmic staining of phospho-Smad3 was observed in non treated MCF-7 cells, whereas predominant nuclear staining BMP7 treatment and in most cells 30 or 60 min after the BMP7 treatment.BMP7 or TGF-βinduced marked increases in the luciferase reporter gene activity that is under the transcriptional control of the Smad3-specific promoter." Human L apoptosis 27696331 Sen_G_0810 BRD7 29117 protein coding BJ -- Aging Accelerate SA-β-gal activity assay//Luciferase reporter assay//Knockdown "BRD7 depletion resulted in a striking increase in proliferation rate in both young and midpassage BJ fibroblasts, suggesting that BRD7 may function in proliferation control before the onset of senescence.BRD7 depletion significantly delays replicative senescence, as these cells continued to Activately proliferate while control firefly luciferase (FF) shRNA expressing cells ceased proliferation and stained positive for senescence-associated (SA) β-galactosidase .Overexpression of BRD7 slows proliferation and results in premature senescence in BJs fibroblasts ." p21//p53 Upregulation//-- qRT-PCR//Western blot "BRD7 depletion in HCT116 cells significantly decreased p21 induction in response to TGF-β, which also occurred in p53 null-HCT116 cells,indicating p53 independence.BRD7 was also required for p21 induction by 1α,25(OH)2D3(vitamin D) in human mammary epithelial cells.BRD7 depletion reduces both the basal and induced expression of p21 in response to p53 activation, although p53 was stabilized as expected,suggesting that BRD7 acts downstream of p53 stabilization. Expression of p21 was also elevated in BRD7-overexpressing cells, providing a mechanistic explanation for their slowed proliferation and premature senescence." -- -- -- -- Human HL apoptosis 20660729 Sen_G_0811 MAPK8 5599 protein coding MCF-7 -- Lung cancer Prevent Cell morphological analysis//SA-β-gal activity assay//Flow cytometry "Cells treated with the JNK inhibitor,SP600125, showed senescence-specific large and flat cellular morphology and stained positively for SA-β-Gal activity.Positive SA-β-Gal activity gradually increased up to 7 days after SP600125 treatment.Analysis of cell cycle distribution revealed that only SP600125-treated cells were arrested in the G2/M phase and that the number of aneuploid cells (>4N) also increased." -- -- -- -- Bcl-2-ROS-DDR Activation Colony formation assay//SA-β-gal activity assay//Western blot//BrdU Assay//Trypan blue staining "When H460 lung carcinoma cells were treated with SP600125, cellular morphology, SA-β-Gal activity and colony-formation ability were compatible with senescence, indicating that this biological process was induced . Loss of phospho-Bcl-2 and phospho-pRB was also detected . Furthermore, ROS generation and DDR were observed along with a decrease in S-phase entry .HEFs transfected with siRNA of JNK or Bcl-2 exhibited SA-β-Gal activity and decreased cell numbers ." Human L apoptosis 19855432 Sen_G_0812 GAS2 2620 protein coding SK-HEP-1 -- Hepatocellular carcinoma Accelerate SA-β-gal activity assay//Flow cytometry "First,flow cytometry analysis revealed a significant elevation of sub-G1cell population upon overexpression of GAS2 .V ector-transfected SK-Hep1 cells were devoid of β-galactosidase (similar to mock-transfected control),whereas GAS2-overexpressed SK-Hep1 cells displayed induction of β-galactosidase." p53//p21 Upregulation//Upregulation Western blot "Quantitative analysis demonstrated that up-regulation of GAS2 increased the number of β-galactosidase-positive cells by almost four-fold,accompanied by elevation of the senescence regulators p53 and p21, as determined by western blot." -- -- -- -- Human HL apoptosis 25925944 Sen_G_0813 FASLG 356 protein coding CRC29 -- Colorectal cancer Accelerate SA-β-gal activity assay//Flow cytometry//Colony formation assay We found that chronic CD95L exposure suppressed the colony-forming potential of five out of nine of these cultures by more than 50% .FACS analysis of PI-stained cells did show a marked increase in the number of cells in G2.We found that CD95L stimulation induced expression of senescence-associated β-galactosi-dase (SA-β-GAL). Caspase Activation Western blot "A time course experiment showed that induction of γH2AX following CD95L stimulation paralleled caspase-8 and caspase-3 activation, starting already 2?h following ligand stimulation." p53 -- Knockdown//SA-β-gal activity assay//Western blot "Knockdown of p53 did not prevent upstream events like caspase-8 and caspase-3 activation, iCAD processing or DNA damage induction. Rather, p53 knockdown increased CD95L-induced DNA damage, which is in line with its function as a ‘guardian of the genome' by activating genes involved in cell cycle arrest and DNA repair.26 Despite the increased DNA damage in CD95L-stimulated p53 knockdown cells, these cells did not enter senescence and largely retained their clone-forming potential ." Human HL apoptosis 28300842 Sen_G_0814 KRAS 3845 protein coding L13 -- Colorectal cancer Prevent SA-β-gal activity assay "By contrast, chronic stimulation of KRAS-deficient C26 cells (L13 cells) caused a pronounced growth inhibition, which was associated with the induction of SA-βGAL." -- -- -- -- -- -- -- -- Human HL apoptosis 28300842 Sen_G_0815 SIRT7 51547 protein coding "NIH-3T3,U2OS" -- Aging Prevent EdU assay//SA-β-gal activity assay "The SIRT7GFP overexpressing cells showed a significant decrease in EdU incorporation indicative of fewer S-phase cells at 14 and 18 h .The control GFP overexpressing cells showed significantly more number of SA-β-gal positive cells,while SIRT7GFP overexpressing cells were mostly SA-β-gal negative." p53 Downregulation Western blot We had noted that SIRT7GFP overexpressing cells when treated with doxorubicin at senescent inducing dose (0.25 mM for 2 h) showed lesser p53 accumulation compared to control. -- -- -- -- Human L apoptosis 25445786 Sen_G_0816 ERBB2 2064 protein coding DLD1 -- Melanoma Prevent SA-β-gal activity assay//qRT-PCR "HER2 ablation exaggerated this damage-induced cellular senescence, which was alleviated by HER2 reconstitution, suggesting that HER2 is responsible for the regulation of damage-induced senescence." TBK1 Downregulation Western blot "HER2 expression markedly inhibited the damage-induced TBK1 activation, indicating a critical role for HER2 in the regulation of DNA sensing during the cellular-damage response . " cGAS-STING Downregulation Western blot HER2 suppressed STING- or TBK1-initiated signalling in a dosedependent manner but failed to suppress the activated IRF3 (5SD). Human L apoptosis 31332347 Sen_G_0817 CDKN2A 1029 protein coding WI-38 -- Aging Accelerate SA-β-gal activity assay//Immunofluorescence "We found that p16 overexpression or H2O2 treatment alone induced senescence in WI-38 cells and the combination of both resulted in an additive effect. Meanwhile, the senescence-associated heterochromatin foci (SAHF) assay confirmed the observations in senescence cell staining. Both 3MeK9H3 and HMGA1, two classic markers of SAHF, localized to the specific heterochromatic foci in cells transfected with p16 and treated with H2O2." -- -- -- -- -- -- -- -- Human L apoptosis 28120917 Sen_G_0818 RELA 5970 protein coding "HCT116,HCT116 p53-/-,MCF-7" -- Cancer Accelerate qRT-PCR//SA-β-gal activity assay//qPCR "Senescence-associated (SA) β-galactosidase positive cells were up to 48.0 and 77.6% of the detected HCT116 p53 wt and HCT116 p53-/- cells, respectively. Using real-time qPCR, we examined the expression 12 senescencerelated genes, and found that in HCT116 cells p15, p21, and Rb were also upregulated in addition to p16.We further examined the senescence associated secretory phenotype (SASP) in MCF7, HCT116 and HCT116 p532/2 cells by real-time qRTPCR.IL-6, IL-8, IL-18, IL-1b, TNF-aCXCL1 and CXCR2 were all greatly induced, suggesting that the p65/S536D indeed has the function of wild type RelA/p65 phosphorylation at Ser536 in regulation of SASP phenotype." -- -- -- -- p16 Upregulation Western blot "In these cells p16 protein levels were elevated , suggesting that p16, not p53 mediates the senescence in HCT116 cells." Human HL apoptosis 26375985 Sen_G_0819 TFEB 7942 protein coding "Primary mice Chondrocyte,Primary human Chondrocyte" -- Osteoarthritis Prevent SA-β-gal activity assay//Western blot "The TBHP-treated chondrocytes exerted higher SA-β-gal activity and p16INK4a protein level relative to the control group, whereas TFEB overexpression significantly prevents this increment." -- -- -- -- -- -- -- -- Human L apoptosis 30154423 Sen_G_0820 BUB1 699 protein coding IMR-90 -- Aging Prevent SA-β-gal activity assay//Cell morphological analysis//Flow cytometry//Knockdown "We found that knockdown of either genes resulted in significant increase of SA-βgal positive cells (p< 0.0001 for all cultures) when compared to EV (EV = 9.6% SD 1.9, shB2= 55.4 SD 7.1, shS2= 74.7% SD 2.86, SEN= 80.2% SD 3.42). Cells depleted of either gene also underwent drastic changes in morphology reminiscent of SEN, such as larger size and flatter morphology.When analyzed by FACS for a senescent-like phenotype (i.e. elevated size and/or AF)30, we observed significantly higher number of cells bearing senescent-like features in BUB1 and SMC1A-depleted cultures relative to EV (shB2: p=0.0013; shS2: p<0.0001), similar to what observed in SEN cultures (SEN: p<0.0001)." -- -- -- -- CDKN2A-Rb1//p53-CDKN1A --//-- IF Cultures depleted of BUB1 and SMC1A were significantly enriched for the frequency of CDKN2A and for CDKN1A positive cells. Human L apoptosis 27731420 Sen_G_0821 SMC1A 8243 protein coding IMR-90 -- Aging Prevent SA-β-gal activity assay//Cell morphological analysis//Flow cytometry//Knockdown "We found that knockdown of either genes resulted in significant increase of SA-βgal positive cells (p< 0.0001 for all cultures) when compared to EV (EV = 9.6% SD 1.9, shB2= 55.4 SD 7.1, shS2= 74.7% SD 2.86, SEN= 80.2% SD 3.42). Cells depleted of either gene also underwent drastic changes in morphology reminiscent of SEN, such as larger size and flatter morphology.When analyzed by FACS for a senescent-like phenotype (i.e. elevated size and/or AF), we observed significantly higher number of cells bearing senescent-like features in BUB1 and SMC1A-depleted cultures relative to EV (shB2: p=0.0013; shS2: p<0.0001), similar to what observed in SEN cultures." -- -- -- -- CDKN2A-Rb1//TP53-CDKN1A --//-- IF Cultures depleted of BUB1 and SMC1A were significantly enriched for the frequency of CDKN2A and for CDKN1A positive cells. Human L apoptosis 27731420 Sen_G_0822 DNAJA4 55466 protein coding HaCaT -- Aging Prevent Flow cytometry//Knockdown//MTS assay "44℃hyperthermia treatment significantly reduced the S phase fraction (P < 0.05) in wild-type HaCaT and NC group cells. By themselves, in the absence of hyperthermia, DNAJA4-deficient cells showed significant reductions in the S phase fraction as compared to both wild-type HaCaT and NC cells (P < 0.05).Results obtained from the MTS assay further substantiated those obtained from flow cytometry, that is,44℃ hyperthermia treated DNAJA4-deficient cells show a significant decrease in cell viability as compared to the other cell groups. Moreover, PDTC pretreatment prior to hyperthermia significantly reduced the apoptosis ratio in wildtype and NC groups, as well as the senescence ratio in DNAJA4-knockout group as compared to hyperthermia treatment alone." -- -- -- -- NF-κB -- qRT-PCR//Western blot The DNAJA4-deficiency group showed increased levels of P65 phosphorylation as compared to the wild-type and NC groups after hyperthermia stimulation. Human L apoptosis 29807809 Sen_G_0823 G6PD 2539 protein coding "HCC,G6PD,HCT116" -- Colorectal cancer Prevent SA-β-gal activity assay//Knockdown//Western blot//Cell proliferation assay "Two non-overlapping shRNAs significantly reduced the expression of G6PD and decreased cell proliferation, which was rescued by overexpression of G6PD. Furthermore, G6PD knockdown significantly increased the number of cells positive for SA-β-GAL (β-galactosidase) staining.the expression of p21, one classic marker for senescence, was upregulated in G6PD knockdown cells and restored to normal level when mouse G6PD was overexpressed. Therefore, suppression of G6PD induced cellular senescence in HCC and HCT116 cells." -- -- -- -- -- -- -- -- Human L apoptosis 24352616 Sen_G_0824 MTDH 92140 protein coding "LNCaP,C4-2B" -- Prostate cancer Prevent SA-β-gal activity assay//Cell proliferation assay Pretreatment with AEG-1-selective siRNA promoted senescence as indicated by increased β-galactosidase activity in LNCaP and C42B cells treated with toxic concentrations of DIM and ring-DIMs.Our results showed a significant inhibition of cell proliferation in LNCaP and C42B cells pretreated with AEG-1 siRNA and then treated with DIM compared to DMSO control cells. -- -- -- -- -- -- -- -- Human L apoptosis 28923415 Sen_G_0825 BMI1 648 protein coding -- Kidney Renal tubulointerstitial injury Prevent SA-β-gal activity assay "E-cadherin-positive renal tubules and the percentage of Ki67 positive renal cells were clearly decreased, whereas the percentages of senescence-associated-β-gal (SA-β-gal) positive area and TUNEL-positive cells were increased significantly in Bmi-1-/- mice, compared with wild-type mice." γ-H2AX//Pchk2//p16//p19//p53//p21 --//--//--//--//--//-- Western blot "Protein expression levels of c-H2A.X, pCHK2, p16, p19, p53, and p21 in kidneys were increased significantly in Bmi-1-/- mice, compared with wild-type mice." -- -- -- -- Human L apoptosis 24915841 Sen_G_0826 PMAIP1 5366 protein coding H460 -- Lungcancer Prevent Cell morphological analysis We further found that NOXAsilenced H460 cells displayed classical senescence morphology with an enlarged and flattened cellular shape when MLN4924-induced apoptosis was blocked; this indicated a conversion of the cell death phenotype from apoptosis to senescence in these cells. -- -- -- -- -- -- -- -- Human L apoptosis 24853380 Sen_G_0827 RBM38 55544 protein coding "SMMC-7721,HepG2" -- Hepatocellular carcinoma Accelerate SA-β-gal activity assay//CCK-8 assay//Colony formation assay "The proportion of cells that were positive for β-galactosidase activity, an indicator of cell senescence, was significantly increased in the HepG2-RBM38-OE (p = 0.0077) and SMMC7721 RBM38-OE cell lines (p = 0.0421) compared to the corresponding controls.Up-regulation of RBM38 led to significantly decreased cell proliferation in HepG2-RBM38-OE cells (p = 0.032) and SMMC7721-RBM38-OE cells (p = 0.044) compared to their corresponding control cells. The colony formation assay showed that when RBM38 was over-expressed, the colony number and size were significantly reduced in HepG2-RBM38-OE (colony number, p = 0.01145; colony size, p = 0.0001) and SMMC7721-RBM38-OE cell lines (colony number, p = 0.0116; colony size, p = 0.0001) when compared to their corresponding control cells ." p53//MDM2 Upregulation//Downregulation Western blot//Luciferase reporter assay "Up-regulation of RBM38 lead to a significant increase in wtp53 and inhibition of mdm2 protein levels compared to their corresponding controls.We found that the luciferase activity for a reporter carrying mdm2 3′-UTR-B and -C was significantly repressed by RBM38 in SMMC7721-RBM38-OE cells. By contrast, the mdm2 3′-UTR-A was not responsive to RBM38. In Hep-G2-RBM38-OE cells, the luciferase activities for reporters carrying mdm2 3′-UTR-A, -B and -C were significantly repressed by RBM38." -- -- -- -- Human L apoptosis 30176896 Sen_G_0828 TFEB 7942 protein coding BEAS-2B Lung Chronic obstructive pulmonary disease Prevent MTS assay "Our data shows that CSE-induced significant increase in ROS-activity is controlled by TFEB-induction, using GEM/FIS.Moreover, TFEBinduction by GEM/FIS also rescues the CSE-mediated decrease in cell viability as shown by the MTS assay.We and others have recently observed that tobacco-smoke/eCV exposure leads to cellular senescence ,via ROS-activation that accelerates lung aging and COPD-emphysema pathogenesis. We demonstrate here that CSE-mediated increase in number of senescent cells is significantly diminished by treatment with TFEB-inducing drugs, GEM/FIS." -- -- -- -- -- -- -- -- Human L apoptosis 27835930 Sen_G_0829 TFDP1 7027 protein coding "HeLa,A549,WI-38,Saos-2" -- Cervical cancer Prevent SA-β-gal activity assay//Knockdown "Based on SA-β-Gal activity after DP1 or DP2 siRNA transfection, HeLa, A549 and WI-38 cells, but not p53-null Saos-2 cells, exhibited an increased percentage of SA-β-Gal-positive cells only upon DP1 depletion. The acute loss o DP2 failed to induce senescence." -- -- -- -- p53//p21 --//-- qRT-PCR//Western blot//Knockdown "In all cells examined, the acute loss of DP1, but not DP2, induced a p21Waf1/Cip1 mRNA level comparable to that in the control RNA-treated cells .The accumulation of p53 and p21Waf1/Cip1 protein was clearly detected in DP1-silenced cells, whereas the knock-down of DP2 alone had no effect on the p53 and p21Waf1/Cip1 protein levels in any of the cells that were examined, compared with the control RNA-transfected cells." Human HL apoptosis 22012588 Sen_G_0830 BRCA1 672 protein coding "HBLpS,MCF-7pS,HBLpS-BR,MCF-7pS-BR,HCC1937,HCC+BR" -- Breast cancer Prevent Western blot//SA-β-gal activity assay//BrdU assay "Accordingly to cytologic data, BRCA1-defective cells showed also a more pronounced induction of the cyclin-dependent kinase inhibitor p21 (CDKN1A) after challenging with MMC, supporting a cell cycle arrest.In fact, treatment with MMC or CDDP resulted in a statistically significant increase in the number of elements showing hallmarks of senescence(enlarged cytoplasm, polynucleation, senescence-associated β-galactosidase positivity, and negativity for BrdUrd incorporation) in BRCA1-defective cells compared with control cells." -- -- -- -- -- -- -- -- Human L apoptosis 19372557 Sen_G_0831 KDR 3791 protein coding U373 MG -- Glioblastoma Prevent SA-β-gal activity assay//Knockdown VEGFR2 gene silencing induced a 4- to 5-fold increase in cells positive for SA-β-Gal (P <0.001). -- -- -- -- -- -- -- -- Human L apoptosis 26420897 Sen_G_0832 SOCS1 8651 protein coding IMR-90 -- Aging Accelerate SA-β-gal activity assay "Expression of SOCS1 inhibited cell growth and induced senescence, characterized by senescence-associated β-galactosidase staining and the accumulation of PML bodies." p53 Activation Western blot//Luciferase reporter assay "Intriguingly, during SOCS1-induced senescence, the increase in p53 levels was accompanied with serine 15 phosphorylation of p53.SOCS1 was not able to activate the p53 reporter alone, but it did increase the activity of added p53 to a similar extent as PML." -- -- -- -- Human HL apoptosis 20005840 Sen_G_0833 MECP2 4204 protein coding MSC -- Aging Prevent SA-β-gal activity assay//Knockdown "We observed signs of senescence in cells treated with Ad‐siRNA‐MECP2, as detected by acid β‐galactosidase, when compared with cells transduced with Ad‐siRNA‐CTRL." -- -- -- -- Rb//p53 --//-- RT-PCR//Western blot "In cells transduced with Ad‐siRNA‐MECP2, we observed an increase in RB gene expression at both the mRNA and protein levels and an increase in RB2/P130 gene expression at only the mRNA level. On in vitro MECP2 down‐regulation, we obtained evidence for significant changes in P53 mRNA levels, but no modification of the protein levels occurred." Human HL apoptosis 20065150 Sen_G_0834 SIRT1 23411 protein coding BJ -- Aging Prevent SA-β-gal activity assay//Western blot Accordingly transfection of specific siRNA oligos independently targeting SIRT1 and SIRT2 significantly decreased expression of SIRT1/2 and induced senescence as shown by increased SA-βgal activity in BJ fibroblasts. Induction of senescence is mediated by DNA damage as evidenced by formation of-H2A.X foci and activation of p53-p21CIP1 pathway. A slight increase in levels of p16INK4A was also detected. -- -- -- -- -- -- -- -- Human L apoptosis 25924011 Sen_G_0835 SIRT2 22933 protein coding BJ -- Aging Prevent SA-β-gal activity assay//Western blot Accordingly transfection of specific siRNA oligos independently targeting SIRT1 and SIRT2 significantly decreased expression of SIRT1/2 and induced senescence as shown by increased SA-βgal activity in BJ fibroblasts. Induction of senescence is mediated by DNA damage as evidenced by formation of-H2A.X foci and activation of p53-p21CIP1 pathway. A slight increase in levels of p16INK4A was also detected. -- -- -- -- -- -- -- -- Human L apoptosis 25924011 Sen_G_0836 RUNX1 861 protein coding AML -- Acute leukemia Prevent SA-β-gal activity assay//Knockdown "After two or more electroporations with RUNX1-CBFA2T1 siRNA, but not with control siRNA, a significant fraction of the Kasumi-1 cells stain positive for β-galactosidase. Cell counting revealed up to 50% of senescent cells, whereas mock or control siRNA treatment caused only a minor increase in senescent cells compared to untreated cells." -- -- -- -- -- -- -- -- Human L apoptosis 15298716 Sen_G_0837 RUNX1T1 862 protein coding AML -- Acute leukemia Prevent SA-β-gal activity assay//Knockdown "After two or more electroporations with RUNX1-CBFA2T1 siRNA, but not with control siRNA, a significant fraction of the Kasumi-1 cells stain positive for β-galactosidase. Cell counting revealed up to 50% of senescent cells, whereas mock or control siRNA treatment caused only a minor increase in senescent cells compared to untreated cells." -- -- -- -- -- -- -- -- Human L apoptosis 15298716 Sen_G_0838 ZEB1 6935 protein coding HUVEC -- Aging Prevent SA-β-gal activity assay//Western blot "Finally, HUVEC depleted of ZEB1 showed an induction of senescence, displaying a higher percentage of SA-β-gal-positive cells , and an increase of p21 protein." -- -- -- -- -- -- -- -- Human L apoptosis 21527937 Sen_G_0839 TP53 7157 protein coding "HN 30,HN 31" -- Squamous cell carcinoma Prevent SA-β-gal activity assay//Western blot "Expression of disruptive TP53 mutations (C176F and E336X) led to decreased SA-β-gal activity compared with empty vector control.metformin decreased clonogenic survival following radiation and increased radiation-induced SA-β-gal activity in HN 31 cells (C176F, disruptive TP53), but had little effect in HN 30 cells (wild type TP53) . Furthermore, after inhibition of wild type p53 expression, metformin was found to potentiate SA-β-gal activity and decrease clonogenic survival." -- -- -- -- -- -- -- -- Human L apoptosis 22090360 Sen_G_0840 ILK 3611 protein coding "Rb116,Y79" -- Human retinoblastoma Prevent SA-β-gal activity assay//Knockdown//Flow cytometry " ILK knockdown significantly increased SA-β-gal activity in Rb116 cells as compared to control. As has been shown previously for Rb-negative retinoblastoma cell lines, a 5-day exposure to QLT-0267 significantly increased the fraction of Y79 cells in G2+M-phase. The percentage of cells in G2+M-phase was 39.6 ± 5% as compared to 24.6 ± 2.1% in vehicle control." Rb -- SA-β-gal activity assay ILK siRNA treatment increased SA-β-gal activity in Rb positive cells but not Rb deficient cells. -- -- -- -- Human L apoptosis 26176204 Sen_G_0841 IL6 3569 protein coding HCT116 -- Tumor Prevent SA-β-gal activity assay//Western blot//Knockdown "Senescence was induced by either gp130 knockdown, IL-6 neutralization,or IL-6 knockdown.inhibition of the IL-6-dependent activation of the JAK1– STAT3 pathway induced senescence following DNA damage." -- -- -- -- JAK1-STAT3 Activation Western blot "To confirm that IL-6 is responsible for the phosphorylation of STAT3 and JAK1, an IL-6 neutralizing antibody (Jo-2) was added to the culture medium 1 day after treatment with doxorubicin.Western blot analysis showed that the Jo-2 antibody blocked the phosphorylation of JAK1 and STAT3 but not of p53 and H2AX.These results suggest that autocrine IL-6 activates the JAK1–STAT3 signaling pathway." Human L apoptosis 22521547 Sen_G_0842 NGF 4803 protein coding "Res 186,Res 259,U87,A172" -- Glioma Accelerate SA-β-gal activity assay "Microscopic analyses indicated that after 72 hr of exposure to NGF, the HGG cells showed an increase in SA‐β‐Gal positive staining compared with control, suggesting the induction of senescence." p27//p21//Cyclin D1//p53//p16 //Rb Upregulation//Upregulation//Upregulation//Upregulation//Upregulation//Downregulation Western blot "In our studies following NGF treatment in A172 cells (expressing p53) we observed p27, p21, cyclin D1, pp53, and p16 enhanced expression and the decrease of phospho‐Rb that directly or indirectly lead to accelerated cell senescence." NF-κB Activation Western blot "In addition, we have found the involvement of NF‐kB in promoting senescence in PLGG cells, in fact the NGF is able to activated NF‐kB signaling after 24 hr of treatment." Human L apoptosis 30417351 Sen_G_0843 TRIM32 22954 protein coding "HCT116 p53+/+,HCT116 p53-/-" -- Tumor Prevent SA-β-gal activity assay//Knockdown "While ectopic TRIM32 reduced doxorubicin-induced senescence in HCT116 p53+/+ but not in p53?/? cells, TRIM32 knockdown induced senescence in HCT116 p53+/+ but not in p53?/? cells." p53 Downregulation Western blot//IP "Western blot assays confirmed that TRIM32 overexpression downregulated p53 in p53+/+ HCT116 and RKO tumors.TRIM32-Flag was co-precipitated by the p53 antibody, and vice versa, indicating that the ectopic TRIM32 interacts with p53 in cells." -- -- -- -- Human L apoptosis 25146927 Sen_G_0844 EPO 2056 protein coding DA3/EPOR -- Leukemia Accelerate SA-β-gal activity assay SAβgal staining was detected in nearly all cells treated with DNR and EPO but not in the untreated control cells. -- -- -- -- -- -- -- -- Human L apoptosis 30622244 Sen_G_0845 HRAS 3265 protein coding HuDE -- Aging Accelerate SA-β-gal activity assay//Cell morphological analysis//Flow cytometry "Results confirmed that many H-RasV12 expressing cells showed premature aging within one day from the transfection.Microscope analysis of cells transfected with H-RasV12 showed a senescent phenotype within one day from the end of the selection, with a flattened cellular morphology, followed by detachment from the adhesion surface. Besides, H-RasV12 transfected cells showed a cell growth arrest that was already evident one day after the end of the selection, compared to cells transfected with the empty vector alone as control. Interestingly, analysis by flow cytometry showed that significant fractions of H-RasV12 expressing cells are still in G0/G1 and S-phase of the cell cycle, despite their arrested state and altered morphology. Indeed, cell cycle analysis showed that transfected cells had a 14.3% reduction of G0/G1 phase cells and a 6.4% increase of G2/M phase cells, in comparison with control cells. Moreover a 5.2% increase of cells in sub-G0 phase was present in H-RasV12 expressing cells compared to the control, suggesting a possible apoptosis induction." GAPDH Downregulation Biochemical assay We observed a significant depletion of GAPDH activity in H-RasV12 transfected cells compared to the control cells. -- -- -- -- Human L apoptosis 23284910 Sen_G_0846 NUDT5 11164 protein coding IMR-90 -- Aging Prevent SA-β-gal activity assay//Western blot "Suppressed NUDT5 expression induced significant rates of senescence within one population doubling, as determined by the senescence-associated β-galactosidase (SA-β-gal) activity.Additionally, an immunoblotting analysis showed that the apoptosis-related proteins p53, p21, and caspase-3 were activated in shNUDT5 cells ." -- -- -- -- -- -- -- -- Human L apoptosis 23581889 Sen_G_0847 PDZD2 23037 protein coding DU 145 -- Prostate cancer Accelerate SA-β-gal activity assay "Furthermore, 108 and 107 M sPDZD2 induced senescence of DU145 cells after 6 days of incubation." p21/WAF1 Upregulation Western blot "Similarly, up-regulation of p21CIP1/WAF1 expression to 2.6-, 2.9- and 3.2-fold were observed after the cells were treated with 109, 108 and 107 M sPDZD2, respectively. " p53 Upregulation Western blot Treatment of MCF-7 cells with 108 and 107 M sPDZD2 for 48 h resulted in 2.4- and 2.2-fold increases in p53. Human L apoptosis 18639375 Sen_G_0848 BCL2L12 83596 protein coding MEF -- Glioblastoma Prevent SA-β-gal activity assay//Flow cytometry "Bcl2L12-expressing MEFs exhibited unabated growth for >35 passages, whereas control cultures showed significantly reduced growth rates or underwent growth arrest with coincidental increase in senescence-associated β-galactosidase (SA-β-Gal) activity ." -- -- -- -- p53 Downregulation Western blot Prompted by the molecular p53 pathway analyses in DNA-damaged MEFs showing reduced p53 accumulation in the presence of Bcl2L12. Human L apoptosis 20837658 Sen_G_0849 HIF1A 3091 protein coding IMR-90 -- Aging Accelerate SA-β-gal activity assay//Flow cytometry//Knockdown//Western blot "The knockdown of HIF-1α also strongly suppressed the conversion of the growth-arrested state in Ni-treated cells into senescence,HIF-1α enhanced protein expression of the CDK inhibitor p21 but moderately diminished upregulation of p53 protein or its Ser15 phosphorylation by Ni. we found that its knockdown helped preserve the expression of the S/G2-specific cyclin A at the midrange of our Ni doses and inhibited upregulation of the CDK inhibitor p27." -- -- -- -- -- -- -- -- Human L apoptosis 28552779 Sen_G_0850 TWIST1 7291 protein coding "H460,A549,H358,H727,Calu-1,Calu-6,H1975" -- Lung cancer Prevent SA-β-gal activity assay//Western blot//Knockdown//Cell morphological analysis//Flow cytometry "Representative photomicrographs (20×) of increased SA-β-gal staining in 3 representative NSCLC cell lines following shRNA mediated TWIST1 knockdown(above).We observed a dramatic activation of latent OIS as demonstrated by positive SA-β-Gal staining and characteristic enlarged, flattened cytoplasm morphology in three KRAS mutant NSCLC cells lines (H460, A549, and H358). These findings were further supported by profound cell cycle arrest in these cell lines. as well as by induction of the senescence markers, p21 and/or p27 in all cell lines examined ." -- -- -- -- -- -- -- -- Human HL apoptosis 23364532 Sen_G_0851 RACK1 10399 protein coding CaSki -- Cervical cancer Prevent SA-β-gal activity assay The number of β-galactosidase-positive cells in control CaSki-vector cells was much higher than RACK1 overexpression CaSki cells. -- -- -- -- -- -- -- -- Human L apoptosis 29048616 Sen_G_0852 POT1 25913 protein coding IMR-90 -- Aging Prevent SA-β-gal activity assay "A premature senescent phenotype was observed in IMR90 cells expressing POT1-RNAi, as assayed by SA-β-Gal staining." TRF2 -- qPCR//Western blot//SA-β-gal activity assay The expression of POT1 decreased the loss of telomeric overhang induced by TRF2ΔBΔM.(B) POT1 blocked the senescent phenotype induced by TRF2ΔBΔM. -- -- -- -- Human L apoptosis 15657433 Sen_G_0853 TERF2 7014 protein coding IMR-90 -- Aging Accelerate SA-β-gal activity assay We found that Myc-TRF2ΔBΔM induced premature senescence 16 days after infection in IMR90 cells. -- -- -- -- -- -- -- -- Human L apoptosis 15657433 Sen_G_0854 TMSB4X 7114 protein coding Nucleus pulposus cell Nucleus pulposus tissue Aging Prevent SA-β-gal activity assay "Furthermore, cell aging and apoptosis were also investigated. In situ SA-β-Gal cell staining was conducted for both control and transfected cells, and the P3 generations of both cell groups were compared. The TB-4 recombinant AAV-transfected cells showed less staining than cells from the control group, which indicated that the transfected cells underwent slower cellular aging." -- -- -- -- -- -- -- -- Human L apoptosis 26021512 Sen_G_0855 PIM1 5292 protein coding hCPC Heart left ventricle Heart failure Prevent SA-β-gal activity assay//Cell morphological analysis "SA β-gal positive cells were least prevalent in Nuc-Pim1 hCPCs, with a ?30.25% decrease (p < 0.001) as compared with Nuc-GFP.Longer, spindle-shaped hCPCs were evident after Nuc-Pim1 modification as measured by the length to width ratio and cell roundness. Concurrently, Nuc-Pim1 modification increased the Hayflick limit of hCPCs as compared with PimWT, prolonging the post-mitotic phenotype as demonstrated by the extended expansion capability of modified cells .PimWT and Mito-Pim1 modification resulted in a reduction of SA β-gal+ cells." p53//p16//NS Downregulation//Downregulation//Upregulation Western blot//qPCR "Gene and protein expression of the cell cycle arrest/senescence marker p53 decreased after Nuc-Pim1 modification with a 1.15-fold reduction of mRNA and a 1.45-fold reduction of protein, as measured by qPCR and immunoblot analysis, respectively.Concurrent down-regulation of the p16 gene and protein expression occurred in Nuc-Pim1 hCPCs as evident by 1.85- and 1.96-fold reductions, respectively. A highly significant increase in NS gene expression resulted from Nuc-Pim1 expression in hCPCs (2.59-fold, p < 0.001) compared with PimWT and Mito-Pim1, as measured by qPCR analysis. Concurrent up-regulation of Ns protein expression was also detected in Nuc-Pim1 hCPCs (2.17-fold, p < 0.01) compared with controls . " -- -- -- -- Human HL apoptosis 25882843 Sen_G_0856 TERF2 7014 protein coding SH-SY5Y -- Neuroblastoma Prevent SA-β-gal activity assay//BrdU assay "DN-TRF2 expression in neuroblastoma cells resulted in a significant reduction in their proliferation as indicated by a decrease of BrdU incorporation, limited growth rate and induction of expression of β-galactosidase." p21//p53 Upregulation//Upregulation Western blot Levels of p21 and p53 were increased by more than 10-fold in neuroblastoma cells expressing DN-TRF2. -- -- -- -- Human L apoptosis 16539655 Sen_G_0857 KDM4B 23030 protein coding "HCT116,SW480 " -- Colorectal cancer Prevent SA-β-gal activity assay//Cell morphological analysis//Flow cytometry "Positive SA-β-gal staining was observed at 72 h post-transfection in about 49% and 20% of JMJD2B-silenced HCT116 and SW480 cells, respectively, but only <2% of the control group were SA-β-gal positive.Compared with the control group, there was a significant increase of HCT116 cells with maximum 4N content at 12?h, and an increase of SW480 cells with maximum 2N content at 24?h after JMJD2B silencing in hypoxia, indicative of G2 and G1 phase arrest, respectively. Morphologically, JMJD2B-silenced cells were larger and appeared flattened (data not shown), which is a feature of senescence (Itahana et al, 2007)." -- -- -- -- STAT3 -- Western blot "Although a slight decrease of total STAT3 was visually observed, p-STAT3 expression significantly decreased in JMJD2B-silenced cells in both normoxia and hypoxia, which was confirmed upon quantification." Human HL apoptosis 24473398 Sen_G_0858 VASH1 22846 protein coding HT29 -- Colorectal cancer Accelerate SA-β-gal activity assay "We found significantly increased SA-β-Gal positive cell populations in HT29 tumor cells after transfection with VASH1-A, indicating the induction of tumor cell senescence." -- -- -- -- -- -- -- -- Human L apoptosis 25797264 Sen_G_0859 CDK2AP1 8099 protein coding HDF -- Aging Prevent SA-β-gal activity assay//Knockdown "We found that 91.7% of HDFs transduced with CDK2AP1-shRNA1 and 70.8% of CDK2AP1-shRNA2 transduced cells displayed appearance of enlarged blue cells in contrast to the control EV transduced cells, which failed to exhibit a detectable blue appearance." p53//p21//BAX//PUMA --//--//--//-- Western blot//qPCR "Indeed we have found that HDFs in which CDK2AP1 was down regulated had a significant increase in p53 protein levels. we found that p21 mRNA levels increased by 2.1 fold in the CDK2AP1-shRNA1 cells and by 1.8 fold in the CDK2AP1-shRNA2 HDFs when compared with EV transduced cells. Western blot analysis further confirmed the increase in p21 protein levels.QPCR analysis indicated that BAX mRNA levels were increased by 1.5 fold in the CDK2AP1-shRNA1 transduced HDFs and by 2.4 fold in the CDK2AP1-shRNA2 when compared with EV transduced cells. Similarly, we observed that PUMA mRNA levels were increased by 2.0 fold in the CDK2AP1-shRNA1 cells and by 3.6 fold in cells transduced with CDK2AP1-shRNA2 when compared with EV cells." -- -- -- -- Human L apoptosis 25785833 Sen_G_0860 PLA2R1 22925 protein coding HMEC -- Breast cancer Accelerate SA-β-gal activity assay//Colony formation assay//Western blot "HMECs ectopically expressing PLA2R1 preferentially underwent proliferation arrest as measured by a colony formation assay, a decrease in the phosphor Ser10 of histone 3 (H3-pSer10), and an increased staining in the senescence-associated-β-galactosidase assay." -- -- -- -- -- -- -- -- Human L apoptosis 23994771 Sen_G_0861 GPC3 2719 protein coding "HuH-7,HepG2" -- Hepatocellular carcinoma Prevent SA-β-gal activity assay//Flow cytometry "Suppression of GPC3 caused a significant increase in G1 peak in HepG2 cells (P =0.04 compared with siRNA-N), together with a decrease in S-phase cells.Transfection with siRNA-GPC3 alone caused marked accumulations of SA-β-Gal in both cell lines." TGF-β2 -- Western blot//Semi-RT-PCR "A member of theTGFBR pathway, TGF-β2 (but not TGF-β1 or TGF-β3), was significantly upregulated by GPC3 suppression in both Huh7 and HepG2 cells.The up-regulation of TGF-β2 by siRNA-GPC3 was independently validated using semiquantitative RT-PCR and Western blot analysis to confirm the negative correlation at both transcript and protein levels, respectively." -- -- -- -- Human HL apoptosis 21847365 Sen_G_0862 TGFB2 7042 protein coding HuH-7 -- Hepatocellular carcinoma Accelerate SA-β-gal activity assay//Flow cytometry "We also observed that addition of rhTGF-β2 induced cellular senescence in both Huh7 and HepG2 cells as shown by enhanced accumulation of SA-β-Gal. Huh7 and HepG2 cells were grown in culture medium supplemented with rhTGF-β2 (1 or 5 ng/ml) for a period of 48 hours before flow cytometry analyses. Similar to the effects of GPC3 suppression, significant increases in the G1 peak , together with a decrease in S- and G2-phase cells were observed in both cell lines compared with control cells not treated with rhTGF-β2 ." Cyclin A//Cyclin D1//Rb//p15INK4b//p21//Bcl-xL//Mcl-1//Bcl-2 Downregulation//Downregulation//Downregulation//Upregulation//Upregulation//Downregulation//Downregulation//Downregulation Western blot "Western blot analysis further showed that rhTGF-β2 decreased the expression of cell cycle regulators cyclin A and cyclin D1, as observed with GPC3 suppression . In addition, phosphorylation of retinoblastoma (Rb) protein was decreased, whereas the expression of p15Ink4b and p21Cip1 were increased in both Huh7 and HepG2 cells, indicating a G1 cell cycle arrest. The addition of TGF-β2 also increased the apoptotic response, demonstrated by the reduced expression of antiapoptotic proteins (Bcl-xL and Mcl-1) in both cell lines, and the reduced expression of Bcl-2 in HepG2 cells only (Huh7 cells do not express Bcl-2)." -- -- -- -- Human HL apoptosis 21847365 Sen_G_0863 ERBB4 2066 protein coding "Saos-2,MG63.2" -- Osteosarcoma Prevent SA-β-gal activity assay//Knockdown "After 3, 5 and 7?days in monolayer culture, knockdown of endogenous HER4 significantly inhibited the proliferation of OS cells. Staining of tumor sections with SA-β-gal showed much higher senescence levels in shHER4 derived xenografts. Cytochemical analysis on SA-β-gal activity revealed that there was a dramatic increase of SA-β-gal-positive cells in shHER4 colonies as compared with scramble shRNA colonies." PERK//p21//p27 --//--//-- Western blot//Knockdown "We detected that the results showed that the senescence-associated protein p-ERK, p21 and p27 levels were increased in cells with HER4 knockdown. " -- -- -- -- Human HL apoptosis 29524631 Sen_G_0864 SOD1 6647 protein coding Mammary epithelial cell Mammary Gland Breast cancer Prevent Western blot//Knockdown//SA-β-gal activity assay//Immunohistochemistry "We found an increase in β-gal staining in the SOD1+/?/ErbB2 cells .Using p53 and p16 as markers of senescence, we found a significant increase in both markers in the SOD1?/?ErbB2 mammary glands. To confirm that senescence was induced in the epithelial cells, we performed immunohistochemistry of p16 and found a significant increase in p16 in the SOD1?/?/ErbB2 mammary glands." -- -- -- -- -- -- -- -- Human L apoptosis 31222103 Sen_G_0865 SIRT1 23411 protein coding A549 -- Idiopathic pulmonary fibrosis Prevent Western blot "We found that Sirt1 overexpression partially rescued miR-34a-induced cellular senescence in A549 cells, as evidenced by reduced expression of PAI-1 and p21 in these cells." -- -- -- -- -- -- -- -- Human L apoptosis 27979858 Sen_G_0866 ZBTB7A 51341 protein coding U87 MG -- Glioma Prevent SA-β-gal activity assay We examined the role of Pokemon in RSVinduced cell senescence by detecting the senescenceassociated β-galactosidase (β-gal) activity. We found that overexpression of Pokemon reduced RSV-induced cell senescence in U87MG cells. -- -- -- -- -- -- -- -- Human L apoptosis 25875864 Sen_G_0867 TP53 7157 protein coding HSC-1 -- Skin cancer Accelerate SA-β-gal activity assay "Clones in which PA treatment induced p53 protein expression showed a Xat and extended morphology such as is commonly observed in senescent cells.we examined whether p53-expressing clones execute senescence-like growth arrest by the expression of a senescence marker, SA-β-gal.A large number of the PAtreated Clone 4 cells stained positive for SA-β-gal at 7 days, while none of the cloned cells not treated with PA were stained blue." -- -- -- -- -- -- -- -- Human L apoptosis 19009304 Sen_G_0868 AKAP4 8852 protein coding "COLO 205 ,HCT116" -- Colorectal cancer Prevent SA-β-gal activity assay//Knockdown//Western blot The percentage of senescent cells was significantly higher in COLO 205 (44.2 %) and HCT 116 cells (48.4 %) when transfected with AKAP4 shRNA3 as compared to NC shRNA staining . AKAP4 ablation also resulted in the upregulation of decoy receptor 2 (DCR2) protein expression which is a marker for cellular senescence. -- -- -- -- -- -- -- -- Human L apoptosis 26590805 Sen_G_0869 POT1 25913 protein coding BGC-823 -- Gastric cancer Prevent SA-β-gal activity assay//Cell morphological analysis//qPCR " After 24 h of culturing, the cells were incubated with β-galactosidase and observed microscopically. In the control cells, only one or two cells showed weak staining. However, after silencing hPOT1 with RNAi, the cells appeared large and flat, some of which were strongly stained with blue. The ratio of senescent cells in blue was 144 ± 12.4/105 (n = 3).The telomere length assay showed that the T/S value = [2Ct (telomere)/2Ct (36B4)] – 1 of the hPOT1 RNAi cells (1.383 ± 0.091) was statistically less than that of the control cells (3.043 ±0.548) (P= 0.007, n= 3), indicating that the loss of hPOT1 in BGC823 cells leads to the shortening of the telomere." -- -- -- -- -- -- -- -- Human HL apoptosis 20517159 Sen_G_0870 PPP1R13L 10848 protein coding "OCCC,OVTOKO,KK" -- Ovarian cancer Prevent SA-β-gal activity assay//Knockdown//Western blot "After 72 hr of lentivirus infection, both OVTOKO and KK cells showed more SA‐β‐Gal staining in iASPP knockdown cells than the scramble control.The expression of p21WAF1/Cip1, an inducer for senescence, also increased in OCCC cells with iASPP silencing." -- -- -- -- -- -- -- -- Human L apoptosis 29663364 Sen_G_0871 BTG1 694 protein coding "HCT-15,HCT116" -- Colorectal cancer Accelerate SA-β-gal activity assay//PI staining "BTG1 overexpression caused G2 arrest in HCT-15 cells (p<0.05), while G1 arrest in HCT-116 cells (p<0.05) by PI staining.A lower mitochondrial membrane potential by JC-1 staining and a higher senescence by β-galactosidase staining were detectable in HCT-116 transfectants than the mock and control, while there was no significant alteration in HCT-15 transfectants." -- -- -- -- -- -- -- -- Human HL apoptosis 27447746 Sen_G_0872 DEK 7913 protein coding "Melanocyte,SK-Mel-28,SK-Mel-94,G-361,UACC-62,UACC-257,MM576" -- Melanoma Prevent SA-β-gal activity assay//Flow cytometry//Cell morphological analysis "In 6 of 12 lines analyzed (SK-Mel-28, SK-Mel-94, G-361, UACC-62, UACC-257, and MM576), DEK depletion led to a progressive cell cycle arrest, eventually resulting in a near complete inhibition of cell proliferation as determined by total cell counting and flow cytometry.The reduction of cells in S phase of the cell cycle was accompanied by cell enlargement, flattening, and vacuolization reminiscent of classic senescent phenotypes . In fact, staining for senescence-associated β-galactosidase activity was positive in DEK-depleted melanoma cells." -- -- -- -- -- -- -- -- Human HL apoptosis 19679545 Sen_G_0873 RHEB 6009 protein coding MEF -- Lymphoma Accelerate SA-β-gal activity assay//Flow cytometry//Western blot//Cell morphological analysis "Rheb expression causes morpho logical changes (data not shown), induction of senescence-associated β-galactosidase (SA β-Gal) ,early growth arrest (Vector vs. Rheb, P < 0.0001),and p16 protein induction ." p53//mTORC1 --//-- SA-β-gal activity assay//Flow cytometry//Western blot "As expected, these effects depended on p53 and did not occur in the context of c-Myc expression.Notably, pharmacologic inhibition of mTORC1 with rapamycin could prevent many phenotypic changes associated with senescence induced by Akt and Rheb and had a partial effect on Ras-induced senescence. " -- -- -- -- Human L apoptosis 18708578 Sen_G_0874 BRAF 673 protein coding Melanocyte -- Melanoma Accelerate SA-β-gal activity assay//Cell morphological analysis Expression of BRAF(V600E) induced senescence in melanocytes and led to an increase in the percentage of senescent cells with the characteristic enlarged cell body and positive staining of senescence-associated β-galactosidase (SA-β-Gal) activity. -- -- -- -- -- -- -- -- Human HL apoptosis 19805117 Sen_G_0875 TYRO3 7301 protein coding Melanocyte -- Melanoma Prevent SA-β-gal activity assay//Colony formation assay "Expression of TYRO3 alone did not have clear effect compared to control virus infected melanocytes. Expression of BRAF(V600E) induced senescence in melanocytes and led to an increase in the percentage of senescent cells with the characteristic enlarged cell body and positive staining of senescence-associated β-galactosidase (SA-β-Gal) activity . However, when TYRO3 was co-expressed with the BRAF(V600E) mutant, primary melanocytes formed colonies in which non-senescent cells dominate the population, as indicated by a strong reduction in the percentage of SA-β-Gal positive cells." -- -- -- -- -- -- -- -- Human HL apoptosis 19805117 Sen_G_0876 TIGAR 57103 protein coding OVCA420 -- Ovarian cancer Prevent SA-β-gal activity assay//EdU assay//Knockdown//Flow cytometry "Within 3?h of the pulse-chase, most of the cells in TIGAR-proficient, scrambled siRNA-transfected cells were progressed into G2/M phase whereas the majority of TIGAR KD cells remained in S-phase. In addition to delay in S-phase, G2/M transition is also affected in TIGAR KD cells because remarkably lower number of phospho-H3-positive M-phase cells were observed in TIGAR KD cells compared to TIGAR-proficient counterparts. we observed an increase in SA-β-gal staining in the TIGAR KD cells and the extent of cells undergoing senescence is inversely correlated with TIGAR expression level. Similar results were observed in A2780 cells." -- -- -- -- -- -- -- -- Human HL apoptosis 31508509 Sen_G_0877 GRB2 2885 protein coding "H1299,SPCA1" -- Lung cancer Prevent SA-β-gal activity assay//Flow cytometry "Up‐regulation of GRB2 effectively counteracted the inhibitory effect of miR‐1258 up‐regulation on H1299 cell proliferation. Similarly, GRB2 down‐regulation in SPCA1 cells reversed the proliferation‐promoting effect of low expression levels of miR‐1258. Moreover, it was found that up‐regulation of GRB2 suppressed cellular senescence and apoptosis, whereas the down‐regulation of GRB2 had an opposite effect." -- -- -- -- -- -- -- -- Human L apoptosis 30069987 Sen_G_0878 ZBTB7A 51341 protein coding "FS090,SW 13 53" -- Chondrosarcoma Prevent SA-β-gal activity assay//Flow cytometry "When compared with wild-type and EGFP-siRNA control cells, LRF-siRNA-expressing cells exhibited a significant accumulation of cells in the G 1 phase, with a corresponding reduction of cells in the S and G 2 /M phases .the percentages of β-galactosidase-positive cells were markedly increased in LRF-depleted FS090 (~50%) and SW1353 (~80%) cells ." p53//p21 --//-- Western blot The western blot results demonstrated that p53 was markedly elevated in LRF-depleted FS090 and SW1353 cells.The expression of p21 was also markedly increased. -- -- -- -- Human HL apoptosis 22847180 Sen_G_0879 TBPL1 9519 protein coding HCT116 -- Colorectal cancer Accelerate SA-β-gal activity assay//Knockdown TLP-depleted cells exhibited resistance to senescence after UV irradiation. p53 -- Western blot//qRT-PCR "The protein levels of both total p53 and Lys-382-acetylated p53, which is an activation marker for p53 transcriptional activity, were elevated within 4 h and decreased around 32–36 h after UV irradiation. Notably, TLP was significantly elevated around 32–36 h, and depletion of TLP accelerated the decrease of both total p53 and Lys-382-acetylated p53 during these time points . TLP-depleted cells clearly showed the reduction of p53 transcriptional activity in the late phase (at 32–48 h) of the UV response." -- -- -- -- Human L apoptosis 28082682 Sen_G_0880 YAP1 10413 protein coding A549 -- Lung cancer Prevent SA-β-gal activity assay YAP plus TEAD completely blocked NCTD-induced senescence enhancement. -- -- -- -- -- -- -- -- Human L apoptosis 27903989 Sen_G_0881 TEAD2 8463 protein coding A549 -- Lung cancer Prevent SA-β-gal activity assay YAP plus TEAD completely blocked NCTD-induced senescence enhancement. -- -- -- -- -- -- -- -- Human L apoptosis 27903989 Sen_G_0882 DDX24 57062 protein coding U2OS -- Cancer Prevent SA-β-gal activity assay//Flow cytometry//Knockdown//Western blot//Cell morphological analysis "43% of the total control cells arrested at G1 phase, and DDX24 knockdown led to the significant increase of G1 population to 62%. In accordance, the reduction of S phase is drastic when DDX24 is depleted from cells.DDX24 siRNA treated cells, under same treatment, did not undergo apoptosis but showed even more predominant G1/S arrest.prolonged culturing of DDX24 siRNA treated U2OS cells ultimately resulted in enlarged and flattened cell shape, the phenotypes typical of senescence45. DDX24 knockdown cells appeared nearly 100% positive for β-galactosidase staining, which proves the occurrence of senescence in cells lacking DDX24 protein." p53 -- Knockdown//Western blot "RNAi mediated knockdown of DDX24 in U2OS cells significantly augmented expression level of p21 and PUMA, the classic p53 targets in regulating cell cycle arrest and apoptosis. Therefore, DDX24 acts as a negative modulator of p53 transcriptional activity. " -- -- -- -- Human L apoptosis 25867071 Sen_G_0883 MAPK1 5594 protein coding "EC109,EC109/CDDP" -- Esophageal cancer Accelerate SA-β-gal activity assay//PI staining//Annexin V binding assay Inhibition of ERK1/2 significantly suppressed cisplatin-induced apoptosis and senescence in EC109 and EC109/CDDP cells. -- -- -- -- -- -- -- -- Human L apoptosis 25236911 Sen_G_0884 POT1 25913 protein coding "HT-1080,IMR-90" -- Aging Prevent Knockdown//SA-β-gal activity assay//Cell morphological analysis "Microscopic examination revealed a fraction of the hPot1 knockdown HT1080 cells with senescent phenotypes (not shown)microscopic examination revealed a fraction of the hPot1 knockdown HT1080 cells with senescent phenotypes (not shown).The Pot1 shRNA elicited a more severe senescent phenotype in these cells; many cells exhibited an enlarged and flattened morphology and stained strongly positive for β-galactosidase (β-gal), a marker of senescence." -- -- -- -- -- -- -- -- Human L apoptosis 15620654 Sen_G_0885 RING1 6015 protein coding "HepG2,HCT116" -- Hepatocellular carcinoma Prevent SA-β-gal activity assay//Flow cytometry "Significant induction of senescence was observed, as 3- to 7-fold more cells were positively stained in the RING1-deficient group showed than in the controls. FACS analysis also indicated that, upon RING1 depletion, increased cell subpopulations were found to retain in G1 phase (increased from 60.0% to 68.4% in HepG2 cells, and from 53.1% to 58.9% in HCT116 cells), suggesting that RING1 deficiency might at least cause partial G1 arrest." p53 -- Knockdown//Western blot "RING1 knockdown decreased the ubiquitination of p53.In a reconstituted in vitro ubiquitination assay that included E1, UbcH6 (E2), ATP and Ub, RING1 was also found to ubiquitinate p53." -- -- -- -- Human HL apoptosis 29187402 Sen_G_0886 RANBP9 10048 protein coding "A549,H460" -- Lung cancer Prevent SA-β-gal activity assay//Knockdown "Silencing of RanBP9 resulted in a significant increase of SA-β-gal positive cells following IR exposure in both RanBP9 stably-silenced A549 and H460 cells, compared to their negative controls, respectively." -- -- -- -- -- -- -- -- Human L apoptosis 26943034 Sen_G_0887 TP53 7157 protein coding Saos-2 -- Cancer Accelerate SA-β-gal activity assay//Flow cytometry//Immunostaining "Bright‐field microscopic images of COE alone, COE + vector and COE + p53 (V138) SKOV‐3 cells at 32 °C and 37 °C showed growth arrest in COE + p53 (V138) at 32 °C only .We examined HP1γ nuclear heterochromatin foci in p53 (V138)‐transfected COE Saos‐2 and SKOV‐3 cells (data not shown) at 32 °C and 37 °C. HP1γ foci were observed only at 32 °C in both the cell lines. We observed β‐galactosidase positive senescence in p53 (V138)‐transfected COE cells at 32 °C. Whereas a significant increase in β‐galactosidase positive cells was seen in p53 (V138)‐transfected cells cultured at 32 °C, the number of Ki‐67 positive cells was low." -- -- -- -- -- -- -- -- Human HL apoptosis 26278998 Sen_G_0888 IFITM3 10410 protein coding "ORL-150,ORL-204" Human OSCC primary tumour Oral cancer Prevent SA-β-gal activity assay//Flow cytometry//Knockdown//Cell morphological analysis "When examining cells that did not undergo cell death following IFITM3 knockdown, we noted that the cell morphology appeared to be flattened with an enlarged cytoplasm as well as an accumulation of vacuoles. This significant increase in senescence-associated βgalactosidase staining indicates that IFITM3 knockdown induces senescence in ORL-150 (48%) and ORL-204 (14.2%) cells compared to the respective NT control cells.By doing so, we indeed observed a significant increase of cells in the G1 phase following IFITM3 knockdown in ORL-150 (86.7%) and ORL-204 (62.5%) cells compared to NT control cells, thus confirming that IFITM3 knockdown induces senescence and accumulation of cells in the G1 cell cycle phase." -- -- -- -- CCND1-CDK4-pRb Upregulation Western blot//Knockdown "We found that the expression of CCND1 was markedly reduced following IFITM3 knockdown in both cell lines. Similarly, we found that the CDK4 expression levels were reduced, but recovered at day 6 post-transfection when IFITM3 levels increased again.The reductions in both CCND1 and CDK4 following IFITM3 knockdown eventually resulted in a reduction in RB phosphorylation (pRB) and to a lesser extent in total RB expression." Human L apoptosis 30949979 Sen_G_0889 SIRT3 23410 protein coding Nucleus pulposus cell Nucleus pulposus tissue Disc degenerative disease Prevent SA-β-gal activity assay//Knockdown//Western blot "The results of the Western blot analysis showed that SIRT3 knockdown augmented the senescence marker protein expression of p16INKa, while SIRT3 overexpression suppressed the p16INKa level under TBHP-induced oxidative stress conditions.Compared to the NPCs transfected with scramble lentivirus, the percentage of SA-β-gal-positive staining cells in the SIRT3 knockdown group was increased, whereas SIRT3 overexpression suppressed the percentage of senescent cells under conditions of oxidative stress." -- -- -- -- -- -- -- -- Human L apoptosis 30420619 Sen_G_0890 BECN1 8678 protein coding U87 -- Glioma Prevent SA-β-gal activity assay//Knockdown//Flow cytometry//PI staining//MTT assay "Beclin-1 knockdown was found to inhibit cell proliferation,apparently, due to induction of cell cycle arrest (in 61% to 84% of total cells) in G1 phase.Beclin-1 knockdown in U87 cells results in blue βgalactosidase staining, which is known to be associated with expression and pH-dependent activation of a lysosomal enzyme senescence-associated β-galactosidase (SA-β-gal), a commonly used biomarker for cellular senescence." -- -- -- -- -- -- -- -- Human HL apoptosis 29991800 Sen_G_0891 KDM2B 84678 protein coding "MDA-MB-231,T47D,MCF-7" -- Breast cancer Prevent SA-β-gal activity assay//Knockdown//Flow cytometry "Flow cytometry of EtBr-stained semiconfluent cell cultures growing under normal tissue culture conditions revealed that the knockdown of NDY1 induces a partial G1 arrest in all the cell lines,and suggested that NDY1 contributes to progression from G1 to S. stained for β-galactosidase (β-gal), revealed that the knockdown elicits a strong senescence phenotype, which, however, is limited to T47D cells." -- -- -- -- -- -- -- -- Human L apoptosis 24853546 Sen_G_0892 TP63 8626 protein coding "keratinocyte,JHU-011,JHU-029" Foreskin Skin cancer Prevent SA-β-gal activity assay//Knockdown//Flow cytometry//Cell morphological analysis "Induction of the cell cycle inhibitor p21CIP1 was observed following knockdown of p63 expression by RNAi in primary human keratinocytes and was associated with an increase in G1-phase cells and a marked decrease in S phase.Little or no evidence of cell death was observed in primary human keratinocytes following lentiviral p63 knockdown. Instead, morphologic features of cellular senescence were observed in a subset of cells, and these changes correlated with staining for SA-β-gal." -- -- -- -- -- -- -- -- Human L apoptosis 17018588 Sen_G_0893 BECN1 8678 protein coding HT22 -- Aging Prevent SA-β-gal activity assay//Western blot "SA-β-gal staining gradually increased in a time-dependent manner when HT22 cells were treated with glutamate or H2O2. Also, levels of some senescence markers including the cyclin-dependent kinase inhibitors p16INK4a, p21, p53 and p27Kip1 increased over time in HT22 cells treated with glutamate or H2O2.Also, treatment of ALLN calpain inhibitor caused a decrease of these senescence markers in 0.5 mM H2O2 -treated HT22 cells." -- -- -- -- -- -- -- -- Human L apoptosis 30807795 Sen_G_0894 EP400 57634 protein coding "U2OS,IMR-90" -- Aging Prevent SA-β-gal activity assay//Western blot//Flow cytometry//qPCR//DAPI staining//Knockdown "We found that p400 knockdown induces the activation of the gene encoding the p21 cell cycle-dependent kinase inhibitor, as well as the concomitant accumulation of cells in the G1 phase of the cell cycle. As already demonstrated [11], transfection of p400 siRNA leads to senescence of IMR90 cells, as observed by the appearance of the so-called SAHF (Senescence-Associated Heterochromatin Foci), the induction of Senescence–Associated β-Galactosidase activity and induction of p16 mRNA expression." ATM -- Immunofluorescence//Knockdown//Flow cytometry "We observed by immunofluorescence staining that p400 knockdown induces ATM phosphorylation both in IMR90 cells and HCT116 cells.We found that the knockdown of p400 leads to an increase of ROS levels (measured by calculating the mean fluorescence from 25,000 cells) detectable 48 hours and 72 hours following siRNA transfection. " -- -- -- -- Human HL apoptosis 20548951 Sen_G_0895 SRC 6714 protein coding HT-1080 -- Aging Prevent SA-β-gal activity assay//Flow cytometry "Surprisingly, the fraction of SA-β-gal–positive cells was greatly reduced in the Src-expressing population, As expected, most of the control cells were arrested in the fraction with 4N DNA content.By contrast, BrdUrd incorporation was seen in the Src population with DNA content between 4N and 8N, suggesting that these cells exit mitosis and then re-replicate their DNA." p21 -- RT-PCR//Western blot "As expected, RT-PCR analysis indicated that p21waf1 accumulated in control cells 36 to 72 hours after drug treatment. Surprisingly, p21waf1 was not significantly expressed in Src-expressing cells." -- -- -- -- Human L apoptosis 16204065 Sen_G_0896 IL22 50616 protein coding HSC Liver Liver disease Accelerate SA-β-gal activity assay//Immunohistochemistry//qRT-PCR//Western blot "Immunohistochemistry staining revealed an increase in the number of SA‐β‐Gal+ cells in livers of Ad‐IL‐22‐treated mice than in the livers of Ad‐GFP‐treated mice, and these cells tended to reside within fibrotic scar tissue.Approximately 60% of HSCs from Ad‐IL‐22‐treated mice were positive for SA‐β‐Gal staining, whereas only 20% of HSCs from Ad‐GFP‐treated mice were positive.IL‐22 treatment increased the number of SA‐β‐Gal+ HSCs, but decreased telomerase activity in HSCs. IL‐22 exposure up‐regulated the expression of several cellular senescence‐associated proteins, including phosphorylated p53 at serine 15 (p‐p53ser15), p53, p21, and SOCS3." STAT3//p53//SOCS3 Activation//Upregulation//Upregulation SA-β-gal activity assay//qRT-PCR//Western blot//Knockdown "STAT3 protein deletion was confirmed by western blotting. IL‐22 treatment up‐regulated SA‐β‐Gal activity and the expression of p‐p53ser15, p53, and p21 in WT HSCs, but not in STAT3?/? HSCs.The expression of Flag‐caSTAT3 was confirmed by western blotting. Infection with Ad‐Flag/caSTAT3 markedly decreased α‐SMA protein expression , but increased SA‐β‐Gal staining in HSCs . Moreover, the introduction of Flag‐caSTAT3 increased the expression of p‐p53ser15, p53, and p21 in HSCs .IL‐22 exposure up‐regulated the expression of SOCS3 mRNA and protein in HSCs.IL‐22 treatment or caSTAT3 overexpression up‐regulated both p53 and SOCS3 proteins.The effects of IL-22 on the signaling pathways in HSCs. IL-22 exposure significantly activated STAT3 in all samples with peak effects observed at 30-60 min. Furthermore, IL-22-dependent STAT3 activation in HSCs was further confirmed by immunostaining for pSTAT3 in the nuclei of HSCs." -- -- -- -- Human L apoptosis 22473749 Sen_G_0897 PRMT5 10419 protein coding GBMNS Brain Glioblastoma Prevent SA-β-gal activity assay//Flow cytometry//Knockdown "PRMT5 depletion increased the G1 population of GBMNS by atleast 30% implicating G1/S cell cycle arrest. Although control cells had undetectable β-gal-positive cells, there were more than 100 β-gal-positive cells per view field in the PRMT5-depleted GBMNS." p53//p21//Rb --//--// -- RT-PCR//Western blot//Knockdown "PRMT5 knockdown increased the relative mRNA expression of p21 and p53. Further we also probed for the expression of Rb, p53 and p21 proteins with or without PRMT5 knockdown .Consistent with senescence phenotype, we found decreased pRb and increased expression of p53 and its target gene p21." -- -- -- -- Human HL apoptosis 27292259 Sen_G_0898 PTEN 5728 protein coding GBMNS Brain Glioblastoma Accelerate SA-β-gal activity assay//Knockdown "We tested the impact of PTEN overexpression in GBMNS on senescence.Similar to the results obtained with PRMT5 knockdown, PTEN overexpressing cells showed a significant increase in β-gal-positive GBMNS." -- -- -- -- -- -- -- -- Human HL apoptosis 27292259 Sen_G_0899 RB1 5925 protein coding MSC -- Retinoblastoma Prevent SA-β-gal activity assay//Flow cytometry "In samples with silenced RB1, the percentage of cells in S-phase is lower than the unirradiated wild type MSCs. In cells with down-regulated RB1, we observed a significant reduction of clones in basal condition vs. controls. This agrees with the high levels of DNA damage and senescence." -- -- -- -- -- -- -- -- Human L apoptosis 27124644 Sen_G_0900 RBL1 5933 protein coding MSC -- Retinoblastoma Prevent SA-β-gal activity assay//Flow cytometry Cells lacking P107 gene expression showed a reduction in S-phase cells only at 48 hours after irradiation. irradiation of wild-type MSCs increased the percentage of senescent cells. A similar pattern was observed in cells lacking RB2 or P107. -- -- -- -- -- -- -- -- Human L apoptosis 27124644 Sen_G_0901 RBL2 5934 protein coding MSC -- Retinoblastoma Accelerate SA-β-gal activity assay//Flow cytometry "Irradiation of wild-type MSCs increased the percentage of senescent cells. A similar pattern was observed in cells lacking RB2 or P107. In detail, the cells with silenced RB2 showed a degree of senescence lower than controls in basal conditions following X-ray treatment." -- -- -- -- -- -- -- -- Human L apoptosis 27124644 Sen_G_0902 GGCT 79017 protein coding PC-3 -- Prostate cancer Prevent SA-β-gal activity assay//Knockdown "Both βgalactosidase staining and β-galactosidase activity measurement confirmed that pro-GA induced senescence, consistent with prior findings that GGCT knockdown induces senescence." -- -- -- -- -- -- -- -- Human L apoptosis 31519583 Sen_G_0903 RASSF1 11186 protein coding A549 Tumor tissue Colorectal cancer Accelerate SA-β-gal activity assay//Flow cytometry//Cell morphological analysis "Conditional RASSF1A expression arrested A549 cells in the G1 phase of the cell cycle.RASSF1A-mediated cell cycle arrest in A549 human lung cancer cells was accompanied by morphologic and biochemical features of sensescence.Moreover, tumors with conditionally expressed RASSF1A exhibited SA-β-gal activity, which serves as an indicator for senescence occurring in vivo, as well as enhanced p21Cip1/Waf1 expression." p21/WAF1 Upregulation Western blot "In agreement with our findings in A549 cells, RASSF1A expression increased endogenous p21Cip1/Waf1 levels in p21Cip1/Waf1-proficient HCT116 cells." -- -- -- -- Human L apoptosis 19223555 Sen_G_0904 CLU 1191 protein coding -- Lung Idiopathic pulmonary fibrosis Prevent Knockdown//Histological staining//Western blot "After 14-days of bleomycin administration, both bleomycin-treated wildtype and CLU?/? mice had increased percent p-H2A.X+ cell clusters, as defined by clusters of 3 or more positive cells, but there were no significant differences between the genotypes. Further, there was no difference in either 8-Oxo-dG intensity or percent p-H2A.X+ cell clusters, 28-days after saline and bleomycin-treatment of wildtype mice. However, there was a significant increase in 8-Oxo-dG staining intensity and p-H2A.X+ cell clusters, in CLU?/? compared to wildtype mice, 28-days after bleomycin-challenge. Finally, exogenously administered Clusterin protein to saline or bleomycin treated wildtype mice did not modulate the number of p-H2A.X+ cell clusters or the percentage of 8-Oxo-dG positive cells in the airways. there was a significant decrease in the expression of msh2, msh6, and ogg1 transcripts in CLU?/? bleomycin-challenged mice compared to CLU?/? saline-control mice. Moreover, there was a significant decrease in msh6 in CLU?/? compared to wildtype mice following bleomycin challenge at Day 28. There was an increase in whole lung p21 and IL1? proteins and KC transcript expression in the bleomycin-challenged CLU?/? compared with their wildtype counterparts at this time point. Finally, there was a significant increase in sftpc transcript expression in wildtype but not CLU?/? mice, 28 days after bleomycin administration." -- -- -- -- -- -- -- -- Human HL apoptosis 29133960 Sen_G_0905 DAXX 1616 protein coding mOSE -- Ovarian cancer Prevent SA-β-gal activity assay//Cell morphological analysis//qRT-PCR "Morphological observations showed that cultured Daxxflox/flox mOSE cells were larger in size with flattened shapes after 15 days in culture, which is a feature of cellular senescence.This showed that 4% of cells hadundergone senescence after 4 days in culture, and >90% of cells were senescent after 15 days in culture. However, senescence (positive staining for SA-β-gal) was barely detectable in immortalized mOSE cells.p16, p21,and p27 mRNAs were highly expressed in Daxxflox/floxmOSE cells after culture for 1 day, whereas these were weakly expressed in immortalized mOSE cells.Senescence occurred rapidly for Daxxflox/floxmOSE cells at 24 h after infection. SA-β-Gal staining showed that about 35% of Daxxflox/floxmOSE cells infected with pAd-GFP-Cre were stained positively vs. about 10% of Daxxflox/floxmOSE cells that were infected with pAd-GFP as a control." -- -- -- -- p53-p21 -- Western blot//Immunofluorescence//qRT-PCR "p21 and p53 accumulated in DAXX-deleted mOSE cells, which suggested that p21/p53 may play an essential role in DAXX deletion-induced senescence. Immunofluorescent staining showed that p27 expression in DAXX-deleted mOSE cells was significantly increased compared with that in control cells. Western blot and real-time PCR analyses showed that the expression of p27 (a cyclin-dependent kinase inhibitor) wa up-regulated in DAXX-deleted cells. However, NOXA and PUMA (p53 inducible genes) mRNA expression increased significantly in DAXX-deleted cells as shown by real-time PCR.DAXX-induced senescence appeared to be largely p21-dependent." Human HL apoptosis 23542781 Sen_G_0906 CSNK2A1 1457 protein coding "A172,U87 MG" -- Glioblastoma Accelerate SA-β-gal activity assay//Flow cytometry "Treatment with Pifithrin-α reversed CK2-I-induced cell cycle arrest. Similar results were obtained in U87 cells also (data not shown). The increase in senescence-positive cells observed in cells treated with CK2-I both in the presence and absence of TNFα, was abrogated in the presence of Pifithrin-α." p53 Upregulation Western blot "A dramatic increase in p53 levels accompanied with an increase in phosphorylation (Ser-15) and acetylation (Lys373, 382) of p53 was observed upon treatment with CK2-Is either alone or in the presence of TNFα. CK2-Is either alone or in combination with TNFα increased mRNA levels of p53-induced pro-apoptotic molecules GADD45β and Noxa." -- -- -- -- Human HL apoptosis 22318540 Sen_G_0907 CSNK2B 1460 protein coding "A172,U87 MG" -- Glioblastoma Accelerate SA-β-gal activity assay//Flow cytometry "Treatment with Pifithrin-α reversed CK2-I-induced cell cycle arrest. Similar results were obtained in U87 cells also (data not shown). The increase in senescence-positive cells observed in cells treated with CK2-I both in the presence and absence of TNFα, was abrogated in the presence of Pifithrin-α." p53 Upregulation Western blot "A dramatic increase in p53 levels accompanied with an increase in phosphorylation (Ser-15) and acetylation (Lys373, 382) of p53 was observed upon treatment with CK2-Is either alone or in the presence of TNFα. CK2-Is either alone or in combination with TNFα increased mRNA levels of p53-induced pro-apoptotic molecules GADD45β and Noxa." -- -- -- -- Human HL apoptosis 22318540 Sen_G_0908 MAP2K7 5609 protein coding MCF-7 -- Breast cancer Prevent SA-β-gal activity assay "Upon treatment with inhibitors of MEK, or its downstream effector ERK, senescence was induced to the same degree as in replication stress response competent cells, suggesting MEK pathway inhibition effectively recovered the ability of cells to undergo oncogene-induced senescence." -- -- -- -- Mdm2-p21 -- Western blot "This result was confirmed by western blot analysis, where ERK inhibition blocked MDM2 phosphorylation and increased p21 levels." Human HL apoptosis 29768207 Sen_G_0909 MAPK1 5594 protein coding MCF-7 -- Breast cancer Prevent SA-β-gal activity assay "Upon treatment with inhibitors of MEK, or its downstream effector ERK, senescence was induced to the same degree as in replication stress response competent cells, suggesting MEK pathway inhibition effectively recovered the ability of cells to undergo oncogene-induced senescence." -- -- -- -- Mdm2-p21 -- Western blot "This result was confirmed by western blot analysis, where ERK inhibition blocked MDM2 phosphorylation and increased p21 levels." Human HL apoptosis 29768207 Sen_G_0910 BRD4 23476 protein coding Mouse Fibroblast cell -- Aging Prevent Southern blot "Treatment with OTX015 caused significant telomere shortening in both human HeLa cells, and mouse CAST/EiJ fibroblasts." -- -- -- -- -- -- -- -- Human HL apoptosis 28854735 Sen_G_0911 HDAC4 9759 protein coding "U87 MG,U251MG" -- Cancer Prevent ELISA//SA-β-gal activity assay//Western blot//Knockdown "RT treatment with 8 Gy significantly and persistently increased SA-β-gal activity,and up-regulated p21WAF1/CIP1but not p27CIP/KIPor p16ink4a protein expression in HDAC4- but not in HDAC6-silenced U87MG and U251MG cells. The role of p21WAF1/CIP1up-regulation in RT-induced HDAC4-silenced GBM cells senescence, was investigated by silencing p21WAF1/CIP1with specific siRNAs. In the presence of silenced HDAC4, p21WAF1/CIP1 knocking-down significantly (p ≤ 0.01) counteracted RT-induced senescence and restored the ability of silenced-HDAC4 GBM cells to form colonies after RT-treatment. p21WAF1/CIP1 siRNA efficiency is reported." -- -- -- -- -- -- -- -- Human L apoptosis 28342984 Sen_G_0912 MYCN 4613 protein coding "HEL,K562" -- Erythroleukemia Prevent SA-β-gal activity assay//Flow cytometry//FCM analysis//qRT-PCR//Knockdown "Suppression of MYCN resulted in increased senescence-associated acidic β-gal staining. Cell cycle analysis showed an increased percentage of cells with G0/G1 phase and a decreased percentage of cells with S phase in the MYCN-knockdown cells. The results showed that depletion of MYCN elevated the P21 expression in HEL cells analysis showed that etoposide treatment resulted in an increase in P21 expression, and MYCN depletion enhanced the etoposide-induced P21 activation in HEL cells. Further comparison analysis showed that overexpression of MYCN inhibited etoposide-mediated P21 activation in HEL and K562 cells." EZH2//p21 Upregulation//Downregulation qPCR//CHIP//Knockdown "The qPCR analysis showed that knockdown of MYCN significantly decreased the expression of EZH2 mRNA . The results showed that knockdown of MYCN led to significantly reduced enrichment of DNA binding in the EZH2 promoter. Further qPCR analysis showed that the expression of MYCN had positive correlation with the expression of EZH2 in the patients with acute erythroleukemia. The results showed that depletion of MYCN elevated the P21 expression in HEL cells. In this study, FCM analysis showed that etoposide treatment resulted in an increase in P21 expression, and MYCN depletion enhanced the etoposide-induced P21 activation in HEL cells . Further comparison analysis showed that overexpression of MYCN inhibited etoposide-mediated P21 activation in HEL and K562 cells." -- -- -- -- Human HL apoptosis 29022893 Sen_G_0913 CDKN2A 1029 protein coding "NP-9,NP-18,NP-29,NP-31" Tumor tissue tissue Pancreatic cancer Accelerate SA-β-gal activity assay//Flow cytometry//Cell morphological analysis "In three of the four cell lines assayed, cells acquired a more refractile cytoplasm and showed a flattened and enlarged morphology, characteristic of arrested or senescent cells.Results from these experiments clearly indicate that in Ad-p16–transduced NP-9 cells, DNA synthesis was stopped, whereas transduced NP-18 cells did not show variations in the percentage of cells in S phase. NP-29 and NP-31 cells did not show such a clear arrest in their cell cycle profile as NP-9 cells when transduced with Ad-p16. However, significant decreases in S phase percentage were observed in 5-BrdU incorporation experiments . Acidic β-gal staining indicated that NP-9, NP-29, and NP-31 cells were committed to a senescent phenotype, as assessed by the increase in their SA-β-gal activity. NP-18 cells did not show any increase in SA-β-gal activity, corroborating the different destination of these cells when p16 is overexpressed." -- -- -- -- -- -- -- -- Human L apoptosis 11687897 Sen_G_0914 KRAS 3845 protein coding "DLD1(KRAS wt/mut),DWT7(wt/-)" -- Lung cancer Accelerate SA-β-gal activity assay//DAPI staining "In an isogenic model, KRAS wild-type cells were sensitive to treatment with erlotinib alone , with the induction of premature cellular senescence accounting for at least some of the effect. In contrast, erlotinib-treated KRAS-mutant cells were resistant to senescence induction." -- -- -- -- -- -- -- -- Human L apoptosis 24648348 Sen_G_0915 SPRY4 81848 protein coding SK-MEL-119NRAS* Tumor tissue tissue Melanoma Accelerate TUNEL assay//SA-β-gal activity assay//Flow cytometry "In vitro analysis of SK-MEL-119NRAS confirmed the increase in apoptosis and SA-β-galactosidase enzyme activity with SPRY4 induction . Compared to control tumors, SPRY4 tumors also harbored reduced Ki67, enhanced tumor cell apoptosis as shown by TUNEL staining and increased SA-β-gal expression. However, upon induction of iBRAF in SK-MEL-119NRAS, there was a significant arrest, as expected, with the control siNTC." -- -- -- -- -- -- -- -- Human HL apoptosis 30651601 Sen_G_0916 HSPA4 3308 protein coding TF1-L1FK clone A5 -- Aging Prevent MTT assay "We observed that AP20187 exerted a proliferative effect that was about ninefold higher in comparison with untreated and KNK437‐treated cells. In AP20187 plus KNK437‐treated cells, the proliferative response was markedly diminished in comparison with AP20187‐treated cells; however; the proliferative response in these cultures was significantly higher (about fourfold) in comparison with KNK437‐treated cells." Akt -- Western blot "We analyzed lysates of A5 cells that were treated with AP20187 and KNK437 and immunoblotted using antibodies to Akt and Erk1/2. Akt was present in abundant levels in AP20187‐stimulated cells. When cells were coincubated with AP20187 and increasing concentrations of KNK437, a dose‐dependent decrease in Akt production was observed. This effect was less acute in comparison with cells treated with KNK437 alone." -- -- -- -- Human HL apoptosis 16076962 Sen_G_0917 HSPH1 10808 protein coding TF1-L1FK clone A5 -- Aging Prevent MTT assay "We observed that AP20187 exerted a proliferative effect that was about ninefold higher in comparison with untreated and KNK437‐treated cells. In AP20187 plus KNK437‐treated cells, the proliferative response was markedly diminished in comparison with AP20187‐treated cells; however; the proliferative response in these cultures was significantly higher (about fourfold) in comparison with KNK437‐treated cells." Akt -- Western blot "We analyzed lysates of A5 cells that were treated with AP20187 and KNK437 and immunoblotted using antibodies to Akt and Erk1/2. Akt was present in abundant levels in AP20187‐stimulated cells. When cells were coincubated with AP20187 and increasing concentrations of KNK437, a dose‐dependent decrease in Akt production was observed. This effect was less acute in comparison with cells treated with KNK437 alone." -- -- -- -- Human HL apoptosis 16076962 Sen_G_0918 NBNC 4683 protein coding Fibroblast -- Nijmegen Breakage Syndrome Prevent SA-β-gal activity assay "When comparing the telomere attrition rate, we foundthat NBS cells showed a higher telomere shortening rate compared to that in normal cells in vitro. For each replication cycle, the telomere shortening rate of NBS cells is around 30bp faster than that of its respective normal counterparts. Consistent with the accelerated telomere shortening, NBS fibroblasts exhibited a significantly higher percentage of cells undergoing senescence compared to normal cells with the same PDLs." -- -- -- -- -- -- -- -- Human L apoptosis 22161642 Sen_G_0919 SRF 6722 protein coding Fibroblast Prostate Prostatic Hyperplasia Prevent SA-β-gal activity assay//Knockdown "After the second and third transfections of SRFsiRNA, a 5- to 8-fold increase of senescent cells was observed, whereas siSCR transfection did not increase the relative number of senescent cells." -- -- -- -- -- -- -- -- Human HL apoptosis 23308381 Sen_G_0920 TP53 7157 protein coding SH-EP -- Neuroblastoma Accelerate SA-β-gal activity assay//Flow cytometry//Knockdown "This arrest appeared to be permanent for p53 wild-type cells, the majority of which stayed on the plates during the whole duration of the experiment. Arrested p53 wild-type cells expressed acidic β-galactosidase, a marker of cellular senescence. In contrast, however, in p53-deficient cells after DNA damage, growth arrest was either temporary (as judged by reappearance of S-phase) or incomplete. Resumption of cell cycle progression was accompanied by gradual accumulation of cells with both sub-G1 and polyploid (>4C) DNA content. Simultaneously, numerous multinucleated cells and cells with multiple micronuclei appeared (data not shown) and the population underwent significant cell loss: By the end of the period of observation, most parts of the plate surface contained no cells. At the same time, in treated populations of p53-deficient cells, we observed formation of multiple colonies of rapidly dividing cells with no traces of senescence-specific β-galactosidase." -- -- -- -- -- -- -- -- Human HL apoptosis 17974978 Sen_G_0921 KRAS 3845 protein coding MEF -- Lung cancer Accelerate SA-β-gal activity assay//Knockdown Senescence-associated β-galactosidase staining was twofold higher in B600E/K12D cells compared with cells expressing one oncogene.Knockdown of either Cdkn2a (encoding p16 and p19) or Cdkn2b (encoding p15) expression restored proliferation of B600E/K12D MEFs. -- -- -- -- -- -- -- -- Human L apoptosis 26028035 Sen_G_0922 BRAF 673 protein coding MEF -- Lung cancer Accelerate SA-β-gal activity assay//Knockdown Senescence-associated β-galactosidase staining was twofold higher in B600E/K12D cells compared with cells expressing one oncogene.Knockdown of either Cdkn2a (encoding p16 and p19) or Cdkn2b (encoding p15) expression restored proliferation of B600E/K12D MEFs. -- -- -- -- -- -- -- -- Human L apoptosis 26028035 Sen_G_0923 TP53 7157 protein coding HCT116 -- Colorectal cancer Accelerate SA-β-gal activity assay//Knockdown HCT116 p53+/+ cells were much more vulnerable to premature senescence induced by ionizing radiation than p53?/? cells.It is apparent that signals secreted by irradiated p53+/+ cells were more effective in inducing senescence in p53?/? bystander cells than were those from p53?/? in their own bystanders. -- -- -- -- -- -- -- -- Human L apoptosis 26099456 Sen_G_0924 IGF1 3479 protein coding MSC Bone Aging Accelerate SA-β-gal activity assay "The senescent (SA-β-gal positive) MSCs showed a blight-blue staining in their central cytoplasm.It is noteworthy that the reduced proliferation, accumulative NCD and marker gene expressions, and the increased senescence were also observed in the void vector transfected MSCs after the selection, indicating that the unexpected cellular senescence may be, at least partly, due to a side effect of puromycin." Ras Activation Western blot "In unselected cultures, no significant changes in activities of Ras(GTP-Ras/Total Ras) were observed in both void vector and IGF-1 transfected cells comparing to the parallel culture; but in the puromycin-selected cultures, the Ras activities in both void vector and IGF-I transfected MSCs were significantly increased with a higher level found in the void vector transfected cells." -- -- -- -- Human L apoptosis 19177843 Sen_G_0925 TINF2 26277 protein coding "TIN2+/+,TIN2+/DC-cond,TIN2+/+ mTR-/-,TIN2+/DCmTR-/-MEF" Embryo Dyskeratosis congenita Prevent SA-β-gal activity assay//Knockdown//FISH "Furthermore, morphological examination and staining for β-galactosidase showed evidence of senescence in a small fraction of the TIN2+/DC Mtr-/- cells.Q-FISH measurements indicated that at a comparable PD, both TIN2+/DC mTR-/-MEFs had sustained greater telomere shortening than the TIN2+/+ mTR-/- MEFs, with the second TIN2+/DC mTR-/- MEF line showing the greatest reduction in mean telomere length." -- -- -- -- -- -- -- -- Human HL telomere attrition 24449270 Sen_G_0926 NHEJ1 79840 protein coding "CD34+,293T" Blood Aging Prevent Knockdown//Telomere length assay//Cell proliferation assay//qRT-PCR "shNHEJ1.2/ CD34t transduced cells showed an inhibition of growth compared with scramble infected cells, as previously observed in 293T cells. These cells also showed increased expression of p21 (CDKN1A) 3-fold in relation with control cells and telomerase activity was reduced by 30%.Expression of several telomerase genes was also inhibited, mainly TERT (40%), TERC (20%), and also the expression of some of the shelterin genes and shelterinassociated factors: TERF1 (40%), TERF2 (20%) and TINF2 (20%) and also CTC1 (25%) and RTEL (30%)." -- -- -- -- -- -- -- -- Human HL telomere attrition 28369633 Sen_G_0927 TINF2 26277 protein coding HeLa-S3 -- Aging Accelerate SA-β-gal activity assay "TIN2-15C, much more than TIN2-13, retarded cell proliferation and induced senescence-associated β-galactosidase." -- -- -- -- -- -- -- -- Human L telomere attrition 18443218 Sen_G_0928 ZNF365 22891 protein coding MEF -- Breast cancer Prevent SA-β-gal activity assay//Knockdown//Telomere length assay//Western blot "Upon knockdown of ZNF365, numerous 53BP1-positive foci appeared, with many localizing to telomeres, and cultures showed robust activation of p53 as well as an early-senescent phenotype. To measure the degree of telomere dysfunction, we determined the anaphase bridge index (ABI) out of total late anaphase. ZNF365-depleted TKO cells exhibited a high ABI, indicating the presence of unseparated sister chromatids or DNA bridges." p53 -- Knockdown//Western blot//SA-β-gal activity assay//FISH "Upon knockdown of ZNF365, numerous 53BP1-positive foci appeared, with many localizing to telomeres, and cultures showed robust activation of p53 as well as an early-senescent phenotype ." -- -- -- -- Human HL telomere attrition 23776040 Sen_G_0929 MDC1 9656 protein coding IMR-90 -- Aging Prevent SA-β-gal activity assay//Knockdown//Western blot "We noted that upon more prolonged culturing [>2 wk], primary human fibroblasts expressing MDC1 shRNAs sh1 or sh2 started proliferating more slowly than the control cells and attained a senescence phenotype, even in the absence of TRF2-DN.in human cells the up-regulation of p53 and p21 in response to TRF2-DN was unaltered by MDC1 knockdown, and primary human fibroblasts underwent senescence within a week of TRF2 inhibition regardless of the level of MDC1." -- -- -- -- ATM -- Knockdown//RT-PCR//Western blot We found that this shRNA [sh3] has extensive sequence identity to the mRNA for the ATM kinase and induced a significant reduction in ATM protein levels. Human L telomere attrition 17158742 Sen_G_0930 DCLRE1B 64858 protein coding IMR-90 -- Aging Prevent SA-β-gal activity assay//Knockdown//Western blot//BrdU assay "The arrested Apollo knockdown cells had a senescent morphology and expressed SA-β-galactosidase, a marker for cellular senescence.In addition, all Apollo shRNAs induced the upregulation of the CDK inhibitor p21, a read-out for p53 activation.Primary human IMR90 fibroblasts with diminished Apollo mRNA levels showed a clear growth defect.Within a week of introduction of Apollo shRNAs, the cells gradually slowed their proliferation and appeared to arrest." -- -- -- -- -- -- -- -- Human L telomere attrition 16730176 Sen_G_0931 RTEL1 51750 protein coding LCL -- Hoyeraal–Hreidarsson syndrome Prevent Southern blot Transduction of healthy LCL (S1) with RTEL11219 caused significant telomere shortening. -- -- -- -- -- -- -- -- Human HL telomere attrition 23959892 Sen_G_0932 TERT 7015 protein coding Fibroblast Skin Dyskeratosis congenita Prevent Northern blot//TRAP assay//qRT-PCR Northern analysis also verified that cells transduced with the TER/eGFP construct expressed a mature TER RNA of approximately 450 bases in length.Expression of TER and TERT together in AD DC fibroblasts brought the level of telomerase to approximately the same as what was observed in normal fibroblasts that expressed TERT alone.Telomere signal was clearly much greater in AD DC or normal cells that expressed TER and TERT together. -- -- -- -- -- -- -- -- Human L telomere attrition 17381549 Sen_G_0933 TOP2A 7153 protein coding "U2OS,HeLa" -- Cancer Prevent TRF assay TOP2a or TOP2b downregulated U2OS cells displayed telomere shortening phenotypes compared to control cells. TIFs Downregulation FISH//Immunostaining Increases in TIFs were observed in shTOP2a and shTOP2b ALT cancer cells but not in telomerase-positive cells. ALT -- Survival curve//Southern blot//FISH//Immunostaining "ICRF-193-treated ALT cells displayed an inhibition of proliferation in a dose-dependent manner. Telomere shortening was also observed in ICRF-193-treated ALT cells, but not in telomerase-positive cells. We next measured the numbers of APBs and TIFs to determine whether the ALT pathway is affected in drug-treated ALT cells. ICRF-193 treatment decreased APBs and increased TIFs in ALT cells. These findings suggest that TOP2s participate in telomere–telomere recombination and that ALT cells are more sensitive to the TOP2 inhibitor than telomerase-positive cancer cells." Human L telomere attrition 25430478 Sen_G_0934 TERF2 7014 protein coding HEp-2 -- Laryngeal cancer Prevent FISH//CCK-8 assay//Knockdown The telomere length of pEGFP-shTRF2-shRAD51- shNBS1-shTERT treated group was distinctly shorter than saline treated group.HEp-2 cell proliferation was inhibited by pEGFP-shTRF2-shRAD51-shNBS1-shTERT. -- -- -- -- -- -- -- -- Human L telomere attrition 27726944 Sen_G_0935 RAD51 5888 protein coding HEp-2 -- Laryngeal cancer Prevent FISH//CCK-8 assay//Knockdown The telomere length of pEGFP-shTRF2-shRAD51- shNBS1-shTERT treated group was distinctly shorter than saline treated group.HEp-2 cell proliferation was inhibited by pEGFP-shTRF2-shRAD51-shNBS1-shTERT. -- -- -- -- -- -- -- -- Human L telomere attrition 27726944 Sen_G_0936 TERF2 7014 protein coding HEp-2 -- Laryngeal cancer Prevent FISH//CCK-8 assay The telomere length of pEGFP-shTRF2-shRAD51- shNBS1-shTERT treated group was distinctly shorter than saline treated group.HEp-2 cell proliferation was inhibited by pEGFP-shTRF2-shRAD51-shNBS1-shTERT. -- -- -- -- -- -- -- -- Human L telomere attrition 27726944 Sen_G_0937 TERT 7015 protein coding HEp-2 -- Laryngeal cancer Prevent FISH//CCK-8 assay The telomere length of pEGFP-shTRF2-shRAD51- shNBS1-shTERT treated group was distinctly shorter than saline treated group.HEp-2 cell proliferation was inhibited by pEGFP-shTRF2-shRAD51-shNBS1-shTERT. -- -- -- -- -- -- -- -- Human L telomere attrition 27726944 Sen_G_0938 LMNA 4000 protein coding Jun-82 -- Hutchinson-Gilford progeria syndrome Accelerate Cell counting//qRT-PCR "Interestingly,a fibroblast line overexpressing wild-type lamin A (82-6 +wt) also exhibited a diminished replicative lifespan,comparable to those expressing mutant forms of lamin A.Telomeres of fibroblasts overexpressing lamin A,either wildtype or mutant, exhibited accelerated rates of shortening as compared to the parental control.When the rates of the telomere loss were plotted against the labeling indices, however, fibroblasts overexpressing wt lamin A showed the fastest rate of telomere loss ." -- -- -- -- -- -- -- -- Human L telomere attrition 17870066 Sen_G_0939 CGGBP1 8545 protein coding 1064Sk -- Aging Prevent SA-β-gal activity assay//PI staining//Flow cytometry//Knockdown "CGGBP1 depletion by CGGBP1 siRNA resulted in DNA damage as demonstrated by an increase in the number of γH2AX foci as compared with control siRNA-treated 1064Sk cells.Proliferation assays starting at 3 wk after first passage posttransfection and stable selection showed strongly reduced cell proliferation in S164A cells .The slow dividing S164A cells acquired the morphology of senescent fibroblasts and a very strong increase in β-galactosidase positivity. Cell cycle analysis showed an accumulation of cells in S-and G2/M-phases in S164A cells, suggesting a block or slow passage through these phases of cell cycle." -- -- -- -- -- -- -- -- Human L telomere attrition 24196442 Sen_G_0940 BMP7 655 protein coding "HeLa,PMC42" -- Cervical cancer Accelerate Telomerase activity assay//Telomere length assay Incubation of human cervical cancer HeLa cells with different concentrations of BMP7 for 48 hours resulted in significant inhibition of telomerase activity.BMP7 induced significant inhibition of telomerase activity in a dose-dependent manner in PMC42 cells.Analyzing the mean telomere length in control and BMP7-treated cells showed that the mean telomere length in the BMP7-treated group could be f25% shorter than that in the controls. hTERT//c-Myc Downregulation//Downregulation qRT-PCR BMP7 induced a significant downregulation of hTERT gene expression in both HeLa and PMC42 cells.We noted that BMP7 also induced a significant down-regulation of c-myc gene expression . -- -- -- -- Human L telomere attrition 19010887 Sen_G_0941 TERF2 7014 protein coding "AG06814,GM00319,AG00780,AG03141" -- Werner syndrome Accelerate Cell morphological analysis//SA-β-gal activity assay "Notably,greater than 80% of Flag-TRF2△B△M-expressing fibroblasts showed a replicative senescence-like phenotype, exhibiting a flattened morphology, and stained positive for SA-β-gal activity." WRN Binding Western blot//SA-β-gal activity assay "Eight days after transduction, 70% of the wild-type WRN-complemented WS fibroblasts expressing Flag-TRF2△Bstained positive for SA-βgal, demonstrating that TRF2△B-induced cellular senescence was reconstituted by the expression of a functional WRN protein. In contrast, overexpression of Flag-TRF2△B failed to increase the percentage of SA-βgal-positive cells in the lines expressing WRN variants lacking helicase, exonuclase, or both activities." p53-p21 Upregulation Western blot "Western blot analysis shows that p53 and its downstream target, p21, are upregulated in normal fibroblasts expressing Flag-TRF2△B△M." Human L telomere attrition 18212065 Sen_G_0942 PRDX1 5052 protein coding HCT116 -- Aging Prevent Knockdown//Southern blot//TRF analysis//Southern hybridization "The MTH1 knockout clone lost 15 base pairs(bp) of telomeric DNA per population doubling (PD),and the PRDX1/MTH1 double-knockout lost 23 bp/PD.On the contrary, disruption of MTH1 or MTH1 and PRDX1 together reduced the elongation rate in 20% O2to 38 and 9–11 bp/PD, respectively. In 40% O2, telomeres shortened in the absence of MTH1, with faster shortening upon codisruption of PRDX1.When grown in the presence of 20% O2, TSQ1 repeat DNA incorporation was reduced in MTH1 knockout cells when compared with wild type, and a further reduction of the TSQ1 repeat DNA was obtained with the MTH1/PRDX1 double-knockout cells." -- -- -- -- -- -- -- -- Human HL telomere attrition 29773556 Sen_G_0943 NUDT1 4521 protein coding HCT116 -- Aging Prevent Knockdown//Southern blot//TRF analysis//Southern hybridization "The MTH1 knockout clone lost 15 base pairs(bp) of telomeric DNA per population doubling (PD),and the PRDX1/MTH1 double-knockout lost 23 bp/PD .On the contrary, disruption of MTH1 or MTH1 and PRDX1 together reduced the elongation rate in 20% O2to 38 and 9–11 bp/PD, respectively. In 40% O2, telomeres shortened in the absence of MTH1, with faster shortening upon codisruption of PRDX1.When grown in the presence of 20% O2, TSQ1 repeat DNA incorporation was reduced in MTH1 knockout cells when compared with wild type, and a further reduction of the TSQ1 repeat DNA was obtained with the MTH1/PRDX1 double-knockout cells." -- -- -- -- -- -- -- -- Human HL telomere attrition 29773556 Sen_G_0944 SMC5 23137 protein coding SUSM1 -- Aging Prevent Knockdown//SA-β-gal activity assay//Cell morphological analysis//Western blot "Knockdown of the SMC5/6 complex in SUSM1 AL T cells caused a progressive increase in the number of SA–β-gal–positive cells over time.This increase correlated with the substantial decrease in telomere lengths in these cells.AL T cells treated with SMC5/6-specific RNAi showed other expected senescence phenotypes, such as morphological changes and an upregulation of the cyclin-dependent kinase inhibitor p21." -- -- -- -- -- -- -- -- Human L telomere attrition 17589526 Sen_G_0945 SMC6 79677 protein coding SUSM1 -- Aging Prevent Knockdown//SA-β-gal activity assay//Cell morphological analysis//Western blot "Knockdown of the SMC5/6 complex in SUSM1 AL T cells caused a progressive increase in the number of SA–β-gal–positive cells over time.This increase correlated with the substantial decrease in telomere lengths in these cells.AL T cells treated with SMC5/6-specific RNAi showed other expected senescence phenotypes, such as morphological changes and an upregulation of the cyclin-dependent kinase inhibitor p21." -- -- -- -- -- -- -- -- Human L telomere attrition 17589526 Sen_G_0946 BTG2 7832 protein coding BJ -- Aging Accelerate Cell counting//SA-β-gal activity assay//Flow cytometry "The shBTG2 cells exhibited increased proliferative potential compared with control cells.Similarly, in Hs68?shp53 fibroblasts that had bypassed senescence as a result of p53 repression,ectopic BTG2 resulted in cell cycle arrest in G1 and β-galactosi-dase staining indicative of senescence." Pin1 Binding Co-IP//Western blot "The levels of both Pin1 and BTG2 protein were elevated in senescent cells compared with young cells and these levels were unaffected by serum.BTG2 coimmunoprecipitated with Pin1 in both young and senescent cells, and the binding between these two proteins was greatly enhanced after serum stimulation." -- -- -- -- Human L telomere attrition 20569234 Sen_G_0947 TP53 7157 protein coding Hs68 -- Aging Accelerate Cell counting//SA-β-gal activity assay//Flow cytometry "As a consequence of sustained p53 inhibition, both BJ and Hs68 cells escaped senescence and grew an additional 10 MPDs compared with control cells expressing pSuper vector .Ectopic expression of p53RR led to the establishment of senescence as measured by the acidic β-galactosidase assay and by the accumulation of cells in the G1 phase of the cell cycle." -- -- -- -- -- -- -- -- Human L telomere attrition 20569234 Sen_G_0948 CDKN1A 1026 protein coding BJ -- Aging Accelerate Cell counting//SA-β-gal activity assay//Flow cytometry The shp21 cells exhibited increased proliferative potential compared with control cells.Ectopic expression of p21 led to the establishment of senescence as measured by the acidic β-galactosidase assay and by the accumulation of cells in the G1 phase of the cell cycle. -- -- -- -- -- -- -- -- Human L telomere attrition 20569234 Sen_G_0949 CHEK2 11200 protein coding "HFF,IMR-90" -- Aging Accelerate Knockdown//SA-β-gal activity assay//BrdU assay "Knockdown of endogenous Chk2 protein led to short-term responses equivalent to those seen with the dominant-negative Chk2, namely reversion to a‘young’ morphology, reduced SA-β-gal expression and increased BrdU incorporation." p21//Rb//Cyclin A Downregulation//--//Upregulation Western blot "We observed a reduction of p21 protein level,and an increase in the hyperphosphorylated form of Rb and in cyclin A expression compared to age-matched control HFF." -- -- -- -- Human L telomere attrition 15192702 Sen_G_0950 BRCA1 672 protein coding HMEC Mammary Gland Breast cancer Prevent SA-β-gal activity assay//Cell morphological analysis "This premature growth arrest (M*) was observed across multiple patient-derived BRCA1mut/﹢HMECs with different BRCA1 mutations and was observed in BRCA1mut/﹢HMECs well . Cells in M* displayed the senescent phenotype, characterized by enlarged, flattened morphology and positive staining for SA-β-galactosidase." Cyclin A //SIRT1 --//-- Western blot Levels of cyclin A were significantly decreased in senescent BRCA1mut/﹢HMECs compared with WT HMECs.Examination of SIRT1 levels in HMECs from BRCA1-mutation carriers revealed significantly reduced protein expression insenescent cells. pRb -- Knockdown "Compared with control, knockdown of pRb in BRCA1mut/﹢HMECs led to an increase in replicative potential, indicating that pRb activity was mediating premature senescence." Human HL telomere attrition 26106036 Sen_G_0951 TERT 7015 protein coding "WS,Fibroblast" -- Werner syndrome Prevent Immunostaining//Western blot//Southern Blot "The expression of hTERT in WS MSCs appears to rescue senescence through reduction of the p16 level (but not of p53/p21) and the DNA damage marker γH2AX.Longer telomere length was found in WS-MSCtert, but not in WS-MSCp53i,suggesting a rescue of the accelerated telomere attrition by telomerase." -- -- -- -- -- -- -- -- Human HL telomere attrition 24749076 Sen_G_0952 TP53 7157 protein coding "WS,Fibroblast" -- Werner syndrome Accelerate SA-β-gal activity assay//Knockdown "As a consequence in MSCs, p53i enhanced their proliferative potential and rescued the premature senescence phenotype without the need for high telomerase activity and long telomere length." -- -- -- -- -- -- -- -- Human HL telomere attrition 24749076 Sen_G_0953 POT1 25913 protein coding "HFF,BJ" Foreskin Aging Prevent SA-β-gal activity assay//Immunostaining//FISH Suppression of hPOT1 in the HFF and BJ cells that had proliferated for the longest time in culture resulted in a 2- to 4-fold greater increase in doubling time as well as a marked induction in senescence-associated h-galactosidase (SA β-gal)staining. We found colocalization of γ-H2AX and telomeric foci in both early- and late-passage cells in which hPOT1 was suppressed. -- -- -- -- -- -- -- -- Human L telomere attrition 18922974 Sen_G_0954 CDK4 1019 protein coding "HMEC,keratinocyte" Mammary Gland Aging Prevent Growth curve assay "Overexpression of Cdk4mock infection in epidermal keratinocytes and HMECs extended their lifespan to 82 and 70 PD, respectively,compared to controls (in the absence of feeder layers 13 and 16 PD." p16//p53//p21 Upregulation//Upregulation//Upregulation Western blot "The expression of p16INK4a was greatly elevated in Cdk4-overexpressing cells cultured in the absence of feeder layers.However, higher levels of p53 and upregulation of p21WAF1 was seen in cells overexpressing Cdk4 compared to uninfected controls." -- -- -- -- Human L telomere attrition 12545164 Sen_G_0955 HLX 3142 protein coding IMR-90 -- Aging Prevent qRT-PCR//Knockdown//Flow cyotmetry "Depletion of INK4a expression rescued the cell cycle arrest triggered by HLX1 knockdown almost completely, indicating that INK4a is a key mediator of the effects of HLX1 on senescence. However, the knockdown of HLX1 still caused a small decline in BrdU incorporation in cells lacking p16INK4a expression, suggesting that HLX1 could also control senescence by additional mechanisms." INK4a -- Knockdown//qRT-PCR "Depletion of INK4a expression rescued the cell cycle arrest triggered by HLX1 knockdown almost completely, indicating that INK4a is a key mediator of the effects of HLX1 on senescence." -- -- -- -- Human L telomere attrition 24067365 Sen_G_0956 TERT 7015 protein coding "DC,Fibroblast" Skin Dyskeratosis congenita Prevent Flow cytometry "TERT expressing DC-1 and normal cells had much higher telomerase activity.TERT-expressing DC cells exhibited a significant 50% decrease in both DHE and MitoSOX oxidation, relative to empty vector treated cells, demonstrating that overexpression of TERT in DC cells decreased steady-state levels of superoxide." -- -- -- -- p53-p21 Downregulation Western blot TERT expression also reduced levels of phospho-p53 in DC cells. Human L telomere attrition 21087144 Sen_G_0957 FUNDC1 139341 protein coding HeLa -- Aging Accelerate SA-β-gal activity assay//Knockdown "To test the effect of FUNDC1-/HSC70-mediated pathway on cell fate, we established a model of senescence by treating HeLa cells with MG132 for 8 h and then incubating them in normal complete medium for a further 3 days. More than 20% of the cells displayed SA-β-gal-positive staining." AMPK Activation Western blot Immunoblotting analysis showed that the AMPK activity and the levels of oxidized proteins in MG132-pulse-treated cells were consistently higher than those in control cells until 3 days after treatment withdrawal. -- -- -- -- Human L mitochondrial dysfunction 30591555 Sen_G_0958 STAT3 6774 protein coding Fibroblast Lung Idiopathic pulmonary fibrosis Accelerate SA-β-gal activity assay//ELISA//Knockdown//Western blot "We found that treatment with STA-21 supressed levels of p21, as measured by immunoblot confirmed by graphed densitometry, and reduced staining of p21 in the nuclei of H2O2-treated fibroblasts.Reduced IL-6 secretion suggests an attenuated secretory profile, and decreased SA-β-Gal content measured using a cytochemical assay." -- -- -- -- -- -- -- -- Human L mitochondrial dysfunction 30608861 Sen_G_0959 CLPP 8192 protein coding "HepG2,HuH-7" -- Decompensated cirrhosis Prevent SA-β-gal activity assay//Flow cytometry//Immunostaining//Western blot "Both HepG2 and Huh7 cells were transfected with either CLPP-GFP or GFP alone and stable cell clones were selected showing more than 90% GFP positivity.Unlike the control cells which showed a pan GFP expression, the CLPP over-expressing cells showed a punctate pattern in the cytoplasm which colocalized with Mitotracker, thereby confirming its mitochondrial localization.The growth kinetics of CLPP over-expressing cells was similar to their control counterpart. When treated with doxorubicin, the CLPP-GFP cells showed almost 50% reduction in SA-β-Gal staining when compared to control GFP cells. This was also accompanied with increased levels of LaminB1 and decreased levels of γH2AX and H3K9Me3 in doxorubicin treated CLPP cells.Senescence is often accompanied by a secretory phenotype which was much subdued in CLPP expressing cells when exposed to doxorubicin." -- -- -- -- -- -- -- -- Human L mitochondrial dysfunction 30878663 Sen_G_0960 ALOX15B 247 protein coding Pca -- Aging Accelerate SA-β-gal activity assay//BrdU assay//Cell morphological analysis "We followed PC3 cells that had been stably transfected with 15-LOX2 or 15-LOX2sv-b for multiple passages. These cells were derived from clonal cultures and enough cells generally became available for analysis only at P4–P5. To our surprise, these cells also showed passage-related phenotypic changes resembling replicative senescence in NHP cells. For instance, most of the early-passage cells were generally small, Activately proliferating, and SA-βgal-negative (not shown). By P6, 10–15% of the stably transfected cells became big and flat, some of which were also SA-βgal + and most of which were BrdU (not shown). With increasing passage, the percentage of big/flat cells also increased in both 15-LOX2- and 15-LOX2sv-b-expressing PC3 cells." -- -- -- -- -- -- -- -- Human L deregulated nutrient sensing 15750631 Sen_G_0961 BCL6 604 protein coding "WI-38,MEF" -- Aging Accelerate SA-β-gal activity assay//Flow cytometry//Cell proliferation assay "Consistent with the ability of p53 to induce a G1 arrest, BCL6-transduced WI-38 cells exhibited an increase in the G1 phase fraction from 74 versus 42% in control cells; and a progressive reduction in their proliferative rate that could not be overcome by increasing serum concentration or passage in culture (data not shown).Also, certain morphological features, such as cytoplasmic vacuoles and enlarged cell size and flattening were observed. These changes were suggestive of cellular senescence, which was further confirmed by detection of pH-dependent -galactosidase levels. Altogether,within 8 days, 37.5% of BCL6-transduced WI-38 cells displayed evidence of senescence as compared with 6% of GFP transduced or untransduced cells." -- -- -- -- p53 Upregulation Knockdown//Western blot//SA-β-gal activity assay//Proliferation assay "Transduction of BCL6 in Tp53-/- MEFs failed to provoke growth arrest. Senescence was largely p53-dependent because no more than 10% of Tp53- /- MEFs became positive for β-galactosidase. Like WI-38 cells, Tp53+/+ MEFs displayed upregulation of Tp53 upon transduction of BCL6. Not only did BCL6-transduced Tp53 -/- MEFs maintain their proliferative potential, but also acquired the ability to form colonies on tissue culture plates in contrast to control transduced cells." Human L deregulated nutrient sensing 18524763 Sen_G_0962 TPP2 7174 protein coding "Fibroblast,PBMC" Skin Evans syndrome Prevent SA-β-gal activity assay//Knockdown "Finally, as described in murine cells,fibroblasts of P1 also showed increased staining with β-galactosidase indicating cellular senescence. proliferation of control T cells was diminished after pretreatment with butabindide." -- -- -- -- -- -- -- -- Human HL immunosenescence 25414442 Sen_G_0963 TLR8 51311 protein coding "MCF-7,M628,PC-3" -- Aging Prevent SA-β-gal activity assay//Knockdown "Knockdown of TLR8,MyD88, and IRAK4 in tumor cells significantly blocked the Poly-G3-mediated reversal of tumor-induced CD4+T-cell senescence. These results confirmed that TLR8 signaling can prevent the induction of T-cell senescence mediated by tumor cells." cAMP Downregulation ELISA "Indeed, Poly-G3 treatment significantly decreased the cAMP levels in tumor cells. Notably, pretreatment of tumor cells with Poly-G3 also markedly decreased the cAMP levels in T cells co-cultured with tumor cells." -- -- -- -- Human L immunosenescence 25231413 Sen_G_0964 TERT 7015 protein coding HCT116 -- Colorectal cancer Prevent SA-β-gal activity assay//Knockdown "In both p53+ and p53? HCT116 cells, RNAi-mediated knockdown of TERT substantially increased the number of cells that stained positively for senescence-associated β-galactosidase activity, indicative of senescence induction." -- -- -- -- p53 -- SA-β-gal activity assay//Knockdown//Flow cytometry "The level of senescence was higher in p53+ HCT116 TERT knockdown cells than in p53? HCT116 TERT knockdown cells, as expected, because p53-directed pathways contribute to senescence [1].In addition, knockdown of TERT increased the percentage of p53? HCT116 cells but not p53+ HCT116 cells in G2/M." Human HL epigenetic alterations 23284306 Sen_G_0965 ETV1 2115 protein coding HCT116 -- Colorectal cancer Prevent SA-β-gal activity assay//Knockdown "Significantly, RNAi-mediated knockdown of ETV1 or ATR also induced senescence." p53 -- SA-β-gal activity assay//Knockdown//Western blot//Flow cytometry "However, following knockdown of ETV1 or ATR, the induction of senescence was much greater in p53? HCT116 cells compared to p53+ HCT116 cells ,consistent with the difference in TERT levels .Notably, a similar preferential increase in the percentage of p53? HCT116 cells in G2/M occurred following knockdown of ETV1 or ATR." -- -- -- -- Human HL epigenetic alterations 23284306 Sen_G_0966 ATR 545 protein coding HCT116 -- Colorectal cancer Prevent SA-β-gal activity assay//Knockdown "Significantly, RNAi-mediated knockdown of ETV1 or ATR also induced senescence." p53 -- SA-β-gal activity assay//Knockdown//Western blot//Flow cytometry "However, following knockdown of ETV1 or ATR, the induction of senescence was much greater in p53? HCT116 cells compared to p53+ HCT116 cells, consistent with the difference in TERT levels.Notably, a similar preferential increase in the percentage of p53? HCT116 cells in G2/M occurred following knockdown of ETV1 or ATR ." -- -- -- -- Human HL epigenetic alterations 23284306 Sen_G_0967 SKP2 6502 protein coding PC-3 -- Prostate cancer Prevent SA-β-gal activity assay "As predicted, a striking increase of β-galactosidase was found in PC3-shSKP2 cells, as compared to that in PC3-scrambled cells, in agreement with literature that SKP2 deficiency induces cellular senescence." JARID1B -- Western blot SKP2 ablation resulted in a striking increase of JARID1B compared to the control. Quantification analysis revealed that JARID1B levels in PC3-shSKP2 cells were 4-fold higher than that in control cells. -- -- -- -- Human HL epigenetic alterations 25596733 Sen_G_0968 TGFB1 7040 protein coding MEF Embryo Aging Accelerate SA-β-gal activity assay//Immunofluorescence "Disruption of TGF-β signaling by E-616452 restored H4K20me3 levels, attenuated SA-β-gal staining and enhanced the Mki67 signals in Suv4-20h-depleted cells." miR-29//H4K20me3 Upregulation//Downregulation Western blot//qRT-PCR "Activation of TGF-β signaling by oxidative stress was accompanied by increased miR-29 accumulation and accelerated reduction in the abundance of total H4K20me3 .Furthermore, inhibition of TGF-β signaling by inhibitor treatment and Smad4 depletion diminished miR-29 expression, attenuated the reduction in Suv4-20h1 and Suv4-20h2 expression and produced partial recovery of H4K20me3." -- -- -- -- Human L epigenetic alterations 29967491 Sen_G_0969 MYSM1 114803 protein coding "C4-2,22Rv1" -- Prostate cancer Accelerate SA-β-gal activity assay//Flow cytometry "Similarly, a significant change of cell cycle distribution was detected in MYSM1-deficient cells. There was a decrease in the percentage of G1-phase cells and an increase in that of S-phase cells, indicating an accelerated progression of cell cycle. Our results showed that MYSM1 downregulation led to decreased proportion of β-gal positive cells." -- -- -- -- -- -- -- -- Human HL epigenetic alterations 31761786 Sen_G_0970 MYC 4609 protein coding "AG00780,AG03141,Fibroblast" -- Werner syndrome Accelerate SA-β-gal activity assay//Flow cytometry "However, within 2–3 passages, ～30%–70% of the WRN–/– cells expressed senescence-associated (SA-) β-galactosidase and lost proliferative capacity. The senescent phenotype in c-myc-transduced WRN–/– cells was also confirmed at the gene expression level by microarray analysis, which demonstrated elevated expression of several genes characteristic of replicative senescence, such as the matrix proteases. WRN–/– cells that were senescent by virtue of MYC overexpression were largely G1-arrested with a small G2 fraction (data not shown)." WRN Binding Flow cytometry WRN–/– cells that were senescent by virtue of MYC overexpression were largely G1-arrested with a small G2 fraction (data not shown). -- -- -- -- Human HL genomic instability 12842909 Sen_G_0971 WRN 7486 protein coding MEF Embryo Werner syndrome Accelerate Giemsa staining "After ～2 mo in culture, several foci emerged among 8 × 106 senescent G5 mTerc-/- Wrn-/- MEFs at a frequency of ～1.4 × 10-5.Under identical culture conditions, foci were not detected in senescent cultures of ～5 × 107 G5 mTerc-/- Wrn+/+ and ～2.5 × 107 G5 mTerc-/- Wrn+/- MEFs." -- -- -- -- -- -- -- -- Human L genomic instability 16264192 Sen_G_0972 TERT 7015 protein coding BJ -- Aging Prevent SA-β-gal activity assay//Flow cytometry "Despite little induction of senescence-associated protein immediately after metal exposure, there was a progressive and dose-dependent increase of β-galactosidase stained cells up to 30 days after metal treatment of hTERT? cells .Preliminary experiments with higher doses of Cr (0.8?μ M) and V (10?μ M) revealed an increase in G2/M in hTERT? cells, but again there was no change in the hTERT+ cells (data not shown)." -- -- -- -- -- -- -- -- Human L genomic instability 16449970 Sen_G_0973 TWIST1 7291 protein coding "NPTX,HPr-1" -- Aging Prevent SA-β-gal activity assay Higher percentage of SA-β-gal-positive cells was found in the Sh-TWIST transfectants (filled columns) compared with the controls (open columns) in response to both agents in a dose-dependent manner. p14 Downregulation Western blot "Reverse transcriptase–polymerase chain reaction analysis showed that the expression of TWIST was also up-regulated at transcription level which was associated with down-regulation of p14 ARF (right panel). We also found that the p14 ARF expression levels were much lower in the TWIST over-expressing cells, either before or after exposure to H 2 O 2 and CP (right panels), compared with the vector control, confirming the negative effect of TWIST on p14 ARF." -- -- -- -- Human L genomic instability 17690110 Sen_G_0974 BCL6 604 protein coding WI-38 -- Aging Accelerate SA-β-gal activity assay//Flow cytometry//Cell morphological analysis "Consistent with the ability of p53 to induce a G1 arrest, BCL6-transduced WI-38 cells exhibited an increase in the G1 phase fraction from 74 versus 42% in control cells; and a progressive reduction in their proliferative rate that could not be overcome by increasing serum concentration or passage in culture (data not shown). Also, certain morphological features, such as cytoplasmic vacuoles and enlarged cell size and flattening were observed. These changes were suggestive of cellular senescence, which was further confirmed by detection of pH-dependent β-galactosidase levels . Altogether, within 8 days, 37.5% of BCL6-transduced WI-38 cells displayed evidence of senescence as compared with 6% of GFP transduced or untransduced cells." p53 Upregulation qPCR//Western blot "However, just as in fibroblasts, BCL6 transduction powerfully induced TP53 mRNA and protein up-regulation as well as P21.p53 protein levels were correspondingly increased in the presence of BCL6." -- -- -- -- Human L genomic instability 18524763 Sen_G_0975 TERT 7015 protein coding RPTEC Kidney Aging Prevent SA-β-gal activity assay//Flow cytometry//Immunostaining "RPTEC/TERT1 cells were propagated as a mass culture because of poor cloning efficiency. They showed growth rates of 0.25 PD/day and a population doubling time of ～96 h, comparable to the normal counterpart, and have now accomplished >90 PDs, corresponding to more than threefold life span extension since normal RPTECs enter growth arrest after 24 ± 3 PDLs.RPTEC/TERT1 cells retained morphology very similar to early-passage cells as well as few SA-β-Gal-positive cells. Interestingly, a considerable fraction of ～20% positive cells was observed also in early-passage cells.γ-H2AX focus formation in RPTEC/TERT1 cells was not reduced to early-passage cell levels, with a number of 7.1 foci per cell." -- -- -- -- -- -- -- -- Human L genomic instability 18715936 Sen_G_0976 SIRT1 23411 protein coding ES -- Aging Prevent FISH "Repair by NHEJ, the prominent DSB repair pathway in G1 and post-mitotic cells, was also reduced in this assay system, but to a lesser extent.H2O2 treatment caused a significant increase in chromosomal aberrations specifically in SIRT1 deficient cells. The frequency of chromatid breaks was comparable between knock-down and control ES cells, but the number of chromosomal fusions, in particular dicentric chromosomes and Robertsonian translocations, was significantly higher in the absence of SIRT1 ." -- -- -- -- -- -- -- -- Human HL genomic instability 19041753 Sen_G_0977 BRCA1 672 protein coding "MCF-7,HCC1937,HCC+BR,HBL-100" -- Aging Prevent SA-β-gal activity assay//Flow cytometry//Cell morphological analysis//BrdU analysis "In fact, treatment with MMC or CDDP resulted in a statistically significant increase in the number of elements showing hallmarks of senescence (enlarged cytoplasm, polynucleation, senescence-associated β-galactosidase positivity, and negativity for BrdUrd incorporation) in BRCA1-defective cells compared with control cells.Accordingly to cytologic data, BRCA1-defective cells showed also a more pronounced induction of the cyclin-dependent kinase inhibitor p21 (CDKN1A) after challenging with MMC, supporting a cell cycle arrest." -- -- -- -- -- -- -- -- Human L genomic instability 19372557 Sen_G_0978 LIN9 286826 protein coding "BJ,MEF" Embryo Aging Prevent SA-β-gal activity assay//Cell morphological analysis "After 10–13 days in culture, LIN9-depleted cells exhibited a large and flat morphology, a phenotype that is commonly observed in senescent cells.Indeed, staining for senescence-associated β-galactosidase activity, a marker of senescent cells, confirmed senescence after loss of LIN9.Two weeks after deletion of LIN9, MEFs became senescent, as previously described ." p16//p21 --//-- Western blot "Immunoblot analysis showed induction of the cell cycle inhibitors p16INK4a and p21Waf1 in LIN9-depleted cells.Similar to what was observed in human cells, loss of LIN9 in two different MEF clones was also accompanied by induction of p16INK4a and p21Waf1." -- -- -- -- Human L genomic instability 21860417 Sen_G_0979 CTSL 1514 protein coding MEF -- Aging Accelerate Western blot "Altogether, our data demonstrate that the upregulation of CTSL activity upon loss of A-type lamins leads to degradation of 53BP1 and defective repair of DNA DSBs by NHEJ.Whereas Lmna-/-fibroblasts presented defects in the fast phase of repair corresponding to NHEJ, depletion of CTSL was sufficient to restore the repair of IR-induced DSBs to a degree that was indistinguishable from that of WT cells." A-type lamins//53BP1//NHEJ --//Downregulation//-- Western blot "Western blots showing that acute depletion of CTSL by lentiviral transduction with an shRNA leads to stabilization of 53BP1 protein in Lmna-/-MEFs.CTSL overexpression led to increased levels of the Activate form of the enzyme and increased CTSL activity, as well as a decrease in 53BP1 levels.Altogether, our data demonstrate that the upregulation of CTSL activity upon loss of A-type lamins leads to degradation of 53BP1 and defective repair of DNA DSBs by NHEJ." -- -- -- -- Human L genomic instability 21750527 Sen_G_0980 SIRT1 23411 protein coding HCT116 -- Aging Prevent Immunofluorescence "SIRT1 depletion led to an increase in partially condensed, disorganized mass of chromosomes that failed to align properly on metaphaseplate in the presence of relatively normal spindles." histone 1(H1)//condensin I complex --//-- Western blot "Protein gel blot analysis from asynchronous cell culture shows the reduction of the chromatin-bound condensin component CAp-D2 and histone H1 in cells treated with SIRt1 siRNA but not control siRNA.The levels of chromatin-bound histione H1 were significantly reduced in cells transfected with anti-SIRT1 siRNA. However, the levels of chromatin-bound CAP-D2, which is a condensin I component, were significantly reduced in cells transfected with anti-SIRT1 siRNA despite unchanged levels in nuclear soluble fraction." -- -- -- -- Human L genomic instability 21636977 Sen_G_0981 MAD2L1 4085 protein coding IMR-90 -- Aging Prevent SA-β-gal activity assay//Cell morphological analysis "Consistent with this observation we scored in IMR90 human fibroblasts with MAD2 depletion roughly 70% of metaphase spreads with prematurely separated sister chromatids.At this time the majority of the human fibroblasts showed enlarged and flattened morphology a typical feature of cellular senescence.Moreover, these IMR90-siMAD2 cells showed nuclei alterations such as binucleate or multinucleated nuclei in 48% of the cells." p21//p14 --//-- RT-PCR//Western blot "Real time RT-PCR revealed a high increase (seven fold) of p21waf1 mRNA in primary human fibroblasts with MAD2 haploinsufficiency when compared to IMR90-siGFP control cells. Also MCF10A-siMAD2 cells showed p21waf1 increased levels but to a lesser extent than those found in IMR90-siMAD2 cells.As expected western blotting confirmed that p21waf1 protein was significantly higher in siMAD2 than in IMR90-siGFP transfected cells. As expected western blotting confirmed that p21waf1 protein was significantly higher in siMAD2 than in IMR90-siGFP transfected cells.At 72 hours post-transfection, p14ARF mRNA was highly increased in IMR90 human fibroblasts .Similarly, western-blotting experiments showed a high increase of p14ARF protein levels in IMR90-siMAD2 cells." p53-p21 -- RT-PCR "When p53 was post-transcriptionally silenced in IMR90-siMAD2 cells, expression level of the p21waf1 gene resulted similar to that present in IMR90-siGFP control cells, as assessed by Real time RT-PCR analysis." Human L genomic instability 22170163 Sen_G_0982 MLH1 4292 protein coding CD34+ -- Aging Prevent qRT-PCR//Immunocytochemistry "A 10-fold decrease in the RQ of MLH1 expression relative to the normalized sample was identified in 2 of the 5 adult CD34+samples.The percentage of CD34+cells with positive staining for MLH1 declined as a function of donor age. Across the donors analyzed, the percentage of CFC with MLH1 expression declined significantly as a function of age." -- -- -- -- -- -- -- -- Human HL genomic instability 22740444 Sen_G_0983 SIRT6 51548 protein coding "Fibroblast,HCA2-HR" -- Aging Prevent Immunocytochemistry SIRT6 overexpression reduces the number of γH2AX foci in presenescent cells. PARP1//Rad51//Rad51C//Rad52//NBS1//SIRT6//CtIP Activation//Upregulation//Upregulation//Upregulation//Upregulation//Upregulation//Upregulation Western blot "In the presence of PJ34, SIRT6 overexpression had no effect on HR,indicating that SIRT6-mediated rescue of HR in aging cells is dependent on PARP1.Western blot showing overexpression of HR-related proteins in HCA2-HR cells." -- -- -- -- Human L genomic instability 22753495 Sen_G_0984 ATM 472 protein coding HPr-1 -- Aging Accelerate Western blot "Examination of cH2AX expression revealed that knockdown of ATM or ATR both suppressed the induction of cH2AX by androgen treatment, suggesting that the androgen-induced DNA damage response was significantly suppressed by ATM/ATR knockdown." CDC25A Downregulation Western blot "Knockdown of ATM, but not ATR, partially recovered the CDC25A protein expression in LNCaP cells, suggesting that downregulation of CDC25A by androgen requires the activation of ATM." -- -- -- -- Human L genomic instability 23272087 Sen_G_0985 RTEL1 51750 protein coding Lymphoma cell line Blood Aging Prevent Southern blot "Growth curves showing the population doublings of the LCLs over time. All LCLs carrying RTEL1 mutations reached a stage of growth arrest (indicated by red “X”). Interestingly, telomeres in LCLs derived from the parents, each carrying a single heterozygous RTEL1 mutation, were also shorter than those of the noncarrier S1 at a PDL of about 35." TRF1 Binding IP "In addition, in a reciprocal experiment using FLAG-tagged TRF1 and TRF2, only FLAG-TRF1 was found to immunoprecipitate RTEL1.None of the mutations significantly affected the interaction of RTEL1 with TRF1." -- -- -- -- Human HL genomic instability 23959892 Sen_G_0986 UHRF1 29128 protein coding Hepatocyte Liver Hepatocellular carcinoma Accelerate SA-β-gal activity assay//qRT-PCR//RNA-seq "However, senescence associated β-galactosidase (SA-β-gal) staining was detected throughout the liver of most UHRF1-GFP High 5 dpf larvae, but not other transgenic lines. Additionally, the DNA in control hepatocytes was evenly distributed throughout the uniformly sized nuclei compared to the large nuclei where DNA resembled senescence associated heterochromatic foci. RNAseq revealed some pro-proliferative genes(ccnd1 and myc) downregulated." tp53//CDKN1A Upregulation//Upregulation RNA-seq//qPCR "RNAseq and qPCR analysis show that tp53 and its target genes, especially cdkn1a, are significantly induced in the liver of 5 dpf UHRF1-GFP High larvae." -- -- -- -- Human HL genomic instability 24486181 Sen_G_0987 SUV39H1 6839 protein coding Fibroblast -- Aging Prevent Western blot//SA-β-gal activity assay "Among these, SUV39H1, a histone methyltransferase that specifically methylates H3K9 and was previously associated with functions in aging or senescence (S4), was downregulated in senescent cells (PD 54), as confirmed by Western blot analysis. Further, the surviving cells in PD 47 cultures treated with increasing concentrations of chaetocin became senescent, suggesting that the inhibition of SUV39H1 was sufficient to induce senescence in low PD cells." p21 -- Western blot//CHIP "p21 protein levels were increased in senescent cells and correlated with the reduced expression levels of SUV39H1.Further, H3K9me3 was less abundant in the promoter region of IL6 in senescent cells when compared to dividing cells,as determined by ChIP, suggesting that reduced SUV39H1 expression levels affect the gene expression profiles of senescent cells." -- -- -- -- Human L genomic instability 25063769 Sen_G_0988 SPRTN 83932 protein coding LCL -- Hepatocellular carcinoma Prevent DNA fiber assay//Western blot//RNA-seq "To further confirm that mutations in SPRTN are the cause of the DNA replication defect, we transfected patients’ cells with WT SPRTN, which almost completely corrected the replication defect and restored cellular proliferation. siRNA-mediated SPRTN depletion severely affected the progression of DNA replication and induced an increased number of stalled forks, newly fired origins and formation of DSBs in the S phase of the cell cycle. Further, short interfering RNA (siRNA)-mediated depletion of SPRTN in HEK293T and U2OS human cell lines also caused chromosomal instability and severe proliferation defects, respectively. sprtn silencing, verified by a GFP reporter assay, led to a substantial increase of γ-H2AX foci, indicating an evolutionarily conserved functional role of SPRTN in the DNA damage response." -- -- -- -- -- -- -- -- Human HL genomic instability 25261934 Sen_G_0989 SDHD 6392 protein coding Sdh4 -- Paraganglioma Prevent Flow cytometry//Immunochemical staining "It was observed that the lifespan of the sdh4DΔ and sdh4DΔ shh4DΔ mutants was reduced compared with the wild-type yeast at day 7. Our results revealed that cellular ROS in the sdh4DΔ shh4DΔ strain was higher than in the sdh4DΔ strain.In our results, the sdh4DΔ shh4DΔ strain showed lower mtDNA integrity and higher mtDNA mutability ." -- -- -- -- -- -- -- -- Human L genomic instability 25328978 Sen_G_0990 SSX2 6757 protein coding "MCF-7-TETSSX2,A375-TET-SSX2,HEK293-TET-SSX2(H293-TET-SSX2)" -- Melanoma Accelerate SA-β-gal activity assay//Immunochemical staining//Cell morphological analysis "HEK293-TET-SSX2 cells exhibited very high background levels of SA-β-gal activity, and no difference was observed upon induction of SSX2 expression.Immunocytochemical analysis of MCF7-TET-SSX2 cells 7 days after induction of SSX2 expression further substantiated their senescent phenotype, as they exhibited a clear reduction in expression of the proliferation marker Ki67 and presence of g-H2AX DSB foci. Long-term growth (more than 7 days) of MCF7-TET-SSX2 cells with SSX2 expression induced a dramatic change in the morphology of the cells, which adapted an irregular and enlarged shape." -- -- -- -- -- -- -- -- Human L genomic instability 25363656 Sen_G_0991 BRCA1 672 protein coding HMEC Mammary Gland Aging Accelerate Western blot//SA-β-gal activity assay "Western blot analysis of p16INK4a, total p53, p53 (Ser15) and p21 levels in WT and BRCA1mut/tHMECs at indicated PDs. M0, stasis, Ag, agonescence(WT HMECs), M*, premature growth arrest (BRCA1mut/tHMECs). Cells in both M* and Ag displayed the senescent phenotype, characterized by enlarged, flattened morphology and positive staining for SA-β-galactosidase." SIRT1 -- SA-β-gal activity assay "Furthermore, knockdown of SIRT1 in WT HMECs resulted in cell-cycle arrest and morphological changes associated with senescence." p53//pRb --//-- Western blot "We found that this proliferative barrier was associated with elevated levels of all components of the p53 signalling pathway (phosphorylated p53 (Ser15), total p53, p21, p27). Collectively, these data indicate that activation of the pRb pathway is the primary mediator of HIS in BRCA1mut/t epithelial cells, and when bypass of HIS is forced (via down-regulation of pRb), it results in the activation of the p53 pathway and accumulation of additional genomic abnormalities." Human HL genomic instability 26106036 Sen_G_0992 PML 5371 protein coding "MRC-5,WI-38" -- Aging Prevent SA-β-gal activity assay//Western blot//Knockdown "Strikingly,PML knockdown caused a dose-dependent decrease in cell proliferation, morphological changes consistent with a senescent phenotype and an increased expression of the senescence-related β-galactosidase enzyme.Moreover, a senescence molecular pathway was activated, as shown by an increased expression of the p53, p16INK4aand p21WAF1proteins in PML-depleted cells with respect to control cells." -- -- -- -- -- -- -- -- Human L genomic instability 26119943 Sen_G_0993 CDH1 999 protein coding Htertrpe -- Aging Prevent SA-β-gal activity assay "We then hypothesized that this E3-Ubiquitin ligase could be activated in S-phase after severe replication stress. Addition of the APC/C inhibitor proTAME (64) during HU treatment restored Cyclin A2 and Cyclin B1 levels, proving the role of APC/C in this process." -- -- -- -- -- -- -- -- Human L genomic instability 26939887 Sen_G_0994 SIRT6 51548 protein coding U2OS -- Aging Prevent SA-β-gal activity assay We detected significantly increased numbers of senescent U2OS cells (over ~20-fold higher than those of control cells) within a week of SIRT6 depletion by lentiviral short hairpin RNAs (shRNAs). H3K18ac -- Western blot "SIRT6 also promoted H3K18ac deacetylation when overexpressed in cells, whereas the mutant SIRT6 H133Y protein did not.Together, these data identify H3K18ac as a new chromatin substrate of SIRT6 and demonstrate a physiologic role of SIRT6 in maintaining total cellular levels of this histone mark." -- -- -- -- Human HL genomic instability 27043296 Sen_G_0995 WSB1 26118 protein coding MEF Embryo Aging Prevent SA-β-gal activity assay//Western blot "Consistent with previous publications, expressing H-RasV12 in MEFs (H-RasV12-MEFs) caused cellular senescence, characterized by enhanced senescence-associated β-galactosidase (SA-β-gal) staining, flattened cell morphology, and upregulation of p53, p21, and p16." ATM Downregulation SA-β-gal activity assay//Colony formation assay "Interestingly, we observed decreased ATM staining and levels in cells overexpressing WSB1.These results led us to hypothesize that WSB1 might negatively regulate A TM levels and that ATM might be a major target of WSB1 in overcoming OIS.We also obtained similar results using ATM+/+ and ATM?/? immortal MEFs. These results suggest that A TM is a major target of WSB1 in inducing cell transformation." -- -- -- -- Human L genomic instability 27958289 Sen_G_0996 DUSP3 1845 protein coding "HeLa,MEWO" -- Aging Prevent SA-β-gal activity assay//Western blot "The results indicated that decreasing endogenous DUSP3 activity per se caused increased cellular senescence of both cell lines after 72 h (20-30%). As expected, cells exposed to 5 Gy of gamma radiation exhibited increased SA-β-Gal staining, which was further significantly increased in the cells subjected to the combined gamma radiation/DUSP3 inhibitor treatment and reached 40-60% in the HeLa and MeWo cells, indicating that these cells tended to undergo senescence in response to pharmacological DUSP3 inhibition." -- -- -- -- -- -- -- -- Human L genomic instability 28389334 Sen_G_0997 PIM1 5292 protein coding "2BS,IMR-90" -- Aging Accelerate SA-β-gal activity assay//CCK-8 assay "In the meanwhile, 2BS cells and IMR90 cells with UHRF1 knockdown exhibited lower cell proliferation rates and reduced EdU incorporation when compared with corresponding control groups. Furthermore, UHRF1 depletion also increased senescenceassociated β-galactosidase (SA-β-gal) activity in both cell lines. All of these data point to the same notion that knockdown of UHRF1 may induce premature senescence in human diploid fibroblasts." UHRF1 Downregulation LC–MS/MS analysis//IP//Western blot "The eluted protein complex was then resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, silver stained and subjected to LC–MS/MS analysis.The identified proteins were listed,including UHRF1, as well as other known interacting proteins of PIM1 such as Hsp9066and SND1,67suggesting that UHRF1 is a potential target of PIM1.Co-immunoprecipitation of UHRF1 followed by western analysis indicated that PIM1 associated with UHRF1 irrespective of whether the PIM1 kinase was wild-type or inActivate mutant." -- -- -- -- Human HL genomic instability 28394343 Sen_G_0998 SIRT7 51547 protein coding "WI-38,hTRT-WI38" -- Aging Prevent SA-β-gal activity assay//Western blot We found that shRNA depletion of SIRT7 from WI38 primary human fibroblasts led to substantial accumulation of senescent cells within days after SIRT7 depletion.Levels of the senescence marker p16 were also increased in the SIRT7-deficient cell cultures (data not shown). SNF2H Upregulation IP "This analysis revealed specific binding of SIRT7 to both the SNF2H and TIP5 subunits of NoRC.Intriguingly, these experiment salso revealed that overexpression of SIRT7 leads to increased SNF2H protein levels." -- -- -- -- Human L genomic instability 29728458 Sen_G_0999 TIMELESS 8914 protein coding 2BS -- Aging Prevent SA-β-gal activity assay//Western blot "The percentage of senescent cells identified by SA-β-gal staining is significantly increased in response to RasG12Vin 2BS human diploid fibroblasts and the percentage of cells with EdU incorporated is decreased.The cellular senescence model was further confirmed by Western Blot analysis of increased expression of p53, p21 and p16." E2F1 Binding IP "Chromatin immunoprecipitation (ChIP) assay was utilized to confirm the binding of E2F1 to the endogenous TIMELESS promoter. Interaction between E2F1 and TIMELESS promoter region was detected by qRT-PCR. As expected, interaction between E2F1 and TIMELESS promoter was observed in young 2BS cells." -- -- -- -- Human HL genomic instability 30100061 Sen_G_1000 TET1 80312 protein coding "H1299,H1975,H226" Lung Lung cancer Prevent SA-β-gal activity assay "Cell morphology (enlarged size and flattened shape) suggested that these cells were undergoing cellular senescence and this was confirmed by analyzing the cells for β-galactosidase activity, a metabolic marker of senescence." p53 Binding IP "Thus, the hypothesis that WT p53 negatively regulates TET1 transcription via direct binding to its promoter was tested with ChIP using a p53-specific antibody. As expected, no enrichment of p53 at its predicted binding site within the TET1 promoter region was detected in H1299 cells characterized by a p53-null mutation. Conversely, 23,800- and 13,500-fold enrichments of p53 at the TET1 promoter were found in the WT p53 cell lines A549 and H2228, respectively." -- -- -- -- Human HL genomic instability 30622117 Sen_G_1001 SETD1A 9739 protein coding "MDA-MB-231,MCF-7,MDA-MB-468,BT-549,HT1299,A549" "Mammary Gland,Lung" Aging Prevent SA-β-gal activity assay//Cell cycle analysis//Knockdown "Remarkably, knockdown of SETD1A(SETD1A-KD) suppresses proliferation and triggers prompt (72h) and massive cellular senescence, with very large cells expressing characteristic ?--galactosidase (?-gal) activity. The cell cycle arrest resulting from SETD1A-KD is consistent with cellular senescence, and it is not associated with apoptosis as seen by the absence of the caspase-3 and PARP cleavage markers of apoptosis, following 3 and 7-days of SETD1A-KD23,24." SKP2 -- Western blot//qPCR//SA-β-gal activity assay Inducible expression of SKP2 in SETD1A-KD cells(using two different shSET1A constructs) effectively reduced the expression of p27 and p21 proteins.Staining for ?-Galactosidase shows that induction of SKP2 partially suppressed the emergence of ?-Gal-positive senescent cells by 50% following SETD1A-KD. -- -- -- -- Human HL genomic instability 31253781 Sen_G_1002 TRPC7 57113 protein coding Keratinocyte Lung Aging Accelerate SA-β-gal activity assay//Western blot//Knockdown "We found that after UVB irradiation, the knockdown of TRPC7 in keratinocytes significantly decreased the percentage of senescent cells, as determined by SA-β-gal staining .Furthermore, the knockdown of TRPC7 inhibited UVB-induced p53 expression in keratinocytes." -- -- -- -- -- -- -- -- Human HL genomic instability 31755176 Sen_G_1003 AGT 183 protein coding VSMC Aorta Atherosclerosis Accelerate SA-β-gal activity assay "SA-β-gal activity was significantly increased in Ang II–treated VSMCs compared with control cells.Ang II also significantly increased the transcriptional activity of p53 compared with that in vehicle-treated cells, and its effect was dose dependent.Consistent with our in vitro data, treatment with Ang II enhanced SA-β-gal activity in the aortas of apoE-deficient mice." -- -- -- -- p53-p21 Upregulation Western blot//Knockdown//SA-β-gal activity assay "Expression of p21 and p53 was elevated in Ang II–treated VSMCs.Western blot analysis revealed that introduction of E6 effectively reduced the level of p53 protein and also markedly reduced the expression of p21, its target protein.Overexpression of p21 significantly increased the number of SA-β-gal positive cells, whereas p21 knockdown with a small interfering RNA (siRNA) system effectively blunted the effect of Ang II on senescence." Human L Others 16908765 Sen_G_1004 AR 367 protein coding DPC Skin Androgenetic alopecia Accelerate SA-β-gal activity assay//Cell morphological analysis//SAHF//Immunostaining//Western blot "Overexpression of the AR increased both percentage of SA-β-Gal–positive cells and cell size and DHT enhanced the AR effects.A quantitative analysis showed that DHT induced SAHF formation in DPCs, and overexpression of the AR reinforced the effects of DHT compared with vector control-transfected cells.Western blot analyses showed that H2AX had been converted to its phosphorylated form (γ-H2AX) in DPCs in response to DHT, and the c-H2AX/total H2AX ratio was further increased in cells overexpressing the AR." p16 Upregulation Western blot "To gain insight into the mechanism of premature senescence induction in DPCs, we focused on the relationship between the AR and p16INK4aprotein, which is known to be involved in premature senescence and is upregulated in balding DPCs." -- -- -- -- Human L Others 24244503 Sen_G_1005 ARPC1B 10095 protein coding "Saos-2,HDF" -- Aging Accelerate Cell morphological analysis//SA-β-gal activity assay "Those cells became flattened and enlarged just like senescent cells as previously reported.＞50% of cells became to express SA-β-gal activity at 9 days after infection with the tTA-encoding virus(After infection with tTA virus, mRNA levels of p41-Arc was dramatically increased ,whereas control cells infected with the ΔE1 control virus showed no activity." -- -- -- -- -- -- -- -- Human L Others 21628992 Sen_G_1006 ASF1A 25842 protein coding WI-38 -- Aging Accelerate Cell morphological analysis//SA-β-gal activity assay//SAHF "HIRA and ASF1a each caused cells to assume markers of senescence, including a large flat morphology, expression of SA-β-gal activity, and macroH2A-containing SAHF.The effect was more pronounced when both proteins were coexpressed." HIRA Binding Co-IP "Endogenous HIRA and ASF1a coimmunoprecipitated from asynchronously growing WI38 cells, confirming that they directly or indirectly associate in cells." -- -- -- -- Human L Others 15621527 Sen_G_1007 BCL6 604 protein coding MEF -- Aging Prevent Survival curve "Importantly, when expressed in primary MEFs of FVB genetic background (at 37°C), BCL6 was very efficient in inhibiting both spontaneous senescence and premature senescence induced by a RASV12oncogene ." Cyclin D1 Upregulation Western blot "The cyclin D1 protein level is up-regulated by BCL6 in three different cell types.This up-regulation of cyclin D1 is at the level of transcription, as cyclin D1 mRNA was also increased by BCL6 in MEFs." -- -- -- -- Human L Others 11914273 Sen_G_1008 BMI1 648 protein coding WI-38 -- Aging Prevent SA-β-gal activity assay "We infected near-senescent cells with pBabe-Bmi-1.Replicative life span increased by ~4 population doublings (PD),relative to control cells.Consistent with these results,Bmi-1 transiently lowered the fraction of cells expressing SA-β-Gal,a marker of the senescent phenotype." p16 Downregulation Western blot "Most cells in Bmi-1-overexpressing cultures did not express p16.Western analysis confirmed that Bmi-1 overexpression rapidly decreased p16 levels in presenes -cent WI-38 cultures;moreover, p16 levels increased modestly when these cultures senesced." pRb -- Cell counting "By contrast, Bmi-1 had no significant effect on the replicative life span of cells expressing E7(pRb eliminated),E6 and E7, or SV40 large T antigen." Human L Others 12482990 Sen_G_1009 BRAF 673 protein coding Melanocyte "Skin,Foreskin" Aging Accelerate BrdU assay//SA-β-gal activity assay//SAHF "Short-term expression of BRAFE600(3–7 days) led to enhanced melanocyte proliferation that was measured by a moderate but reproducible increase in 5-bromodeoxyuridine (BrdU) incorporation. However, this effect was transient—sustained expression of BRAFE600resulted in marked cell cycle arrest.In most (roughly 80%) of the transduced melanocytes, this was associated with an intense activity of SA-β-Gal, a marker for senescent or stressed cultured cells as well as for aged tissues in vivo6,15.We observed that low levels of BRAFE600also induced SAHF." p16 Upregulation Immunostaining We found that BRAFE600-expressing melanocytes had elevated levels of p16INK4a. -- -- -- -- Human L Others 16079850 Sen_G_1010 CAV1 857 protein coding Fibroblast Foreskin Aging Prevent Western blot//Cell morphological analysis "We observed that a simple reduction in the caveolin-1 status, as determined by the siRNA method, induced in senescent cells morphological changes corresponding to the young cell-like shape, i.e. a small and spindle shape." FAK//Rac1//Cdc42 --//Activation//Activation Western blot//Cell morphological analysis//IP The phosphorylation of FAK was dramatically reduced by the down-regulation of caveolin-1 in caveolin siRNA-transfected senescent cells versus the control firefly luciferase siRNA-transfected senescent cells.Reductions of focal adhesion and actin stress fiber formation via the inactivation of FAK by caveolin-1 status reduction resulted in morphological alterations toward the young cell phenotype.Rho GTPases were recruited into caveolin fractions and were directly linked with caveolin-1 in senescent HDF cells by immunoprecipitation analysis. -- -- -- -- Human L Others 15263006 Sen_G_1011 CBX8 57332 protein coding "MEF,Fibroblast" -- Aging Prevent Flow cytometry//BrdU assay//Colony formation assay "We found that inhibition of CBX8 expression dramatically reduced the S-phase fraction of cells and led to a complete growth arrest.Overexpression of CBX8 bypassed senescence, and the growth rate of the CBX8 expressing cells did not change within the time frame of the experiment.We found that Cbx8 downregulation led to a significant reduction in the number of colonies in all the genetic backgrounds tested." BMI1//INK4a//ARF Binding//Downregulation//Downregulation Western blot//IP//qRT-PCR "Western blot (WB) analysis of protein extracts before depletion (BD) and after depletion (AD) showed a reduction of about 60% of total CBX8 after BMI1 depletion and a reduction of about 20% of total BMI1 after CBX8 depletion.Consistent with binding to the Ink4a-Arf locus, CBX8 expression led to a decrease in Ink4a and Arf mRNA levels." -- -- -- -- Human HL Others 17332741 Sen_G_1012 IL1A 3552 protein coding endothelial cell -- Aging Prevent Cell counting//Cell morphological analysis The daily addition of the IL-lot antisense oligomer to populationsofhuman endothelial cells at 20 population doublings resulted in asignificant extension of cell proliferation .Removal of the IL-la antisense oligomer from a cell population after an extended number of population doublings resulted in a reduction in the proliferative capacity of the monolayer and the generation of the senescent cell phenotype. -- -- -- -- -- -- -- -- Human L Others 2218499 Sen_G_1013 TGFB1I1 7041 protein coding "KMST-6,SUSM1" -- Aging Accelerate Colony formation assay//Cell morphological analysis "The number of colonies decreased as the ratio of CMV/S5 to CMV in the transfected plasmid mixture increased, suggesting that the forced expression of the hic-5 cDNA inhibited the growth of these immortalized human cells. Enlarged, flattened cells in a senescence-like state appeared and constituted about 20% of the population over 30 PD." -- -- -- -- -- -- -- -- Human L Others 9032249 Sen_G_1014 HRAS 3265 protein coding IMR-90 -- Aging Accelerate Western blot//SA-β-gal activity assay "As expected,vector-transduced cells transiently induced c-fos 30 min after restimulation.No c-fos message was detected, however, in H-rasV12-transduced cells.H-rasV12-transduced IMR90 cells displayed a significant accumulation of PAI-1 mRNA,compared with vector-transduced cells (2.5-fold large tran script; 7.5-fold small transcript).The percentage of SA-β-gal positive cells in H-rasV12-expressing populations was initially low, but increased at day 3 and reached 60% by day 6." -- -- -- -- p53//p16-Rb --//-- BrdU assay "In contrast to wild-type MEFs, p53-/-or p16-/-MEFs continued to incorporate BrdU and proliferate following introduction of H-rasV12." Human L Others 9054499 Sen_G_1015 VEGFA 7422 protein coding HDMEC Foreskin Aging Prevent Cell morphological analysis//Cell counting//SA-β-gal activity assay "When cultures were supplemented with a single pulse of VPF/VEGF at a final concentration of 5 ng/ml at the time of each passage after PD26, many HDMECs continued to maintain a relatively small size even beyond PD56 and continued to proliferate.When VPF/VEGF was withdrawn HDMEC developed a senescent morphology within 7 days. Addition of VPF/VEGF to senescent PD36 HDMEC induced renewed cell proliferation and restored a more normal morphology.senescent cells subsequently cultured with VPF/VEGF reduced their expression of neutral β-galactosidase." p21//p16//p27 Downregulation//Downregulation//Downregulation Western blot p16 and p21 were only weakly expressed in early passage HDMEC(lane 3)but both were strongly expressed in senescent cells (lane 2). The expression of both was markedly reduced when senescence was prevented by culture with VPF/VEGF (lane 4).p27/Kip1 was moderately expressed in senescent HDMEC but at very low or undetec -table levels in both early passage cells cultured without VPF/VEGF and in late passage cells cultured with VPF/VEGF. -- -- -- -- Human L Others 9160882 Sen_G_1016 CDKN1A 1026 protein coding LF1 -- Aging Accelerate Southern hybridization analysis Southern (DNA) hybridization revealed that the apparent extension of life-span was accompanied by loss of heterozygosity (LOH) at the p21 locus. -- -- -- -- -- -- -- -- Human L Others 9242615 Sen_G_1017 TP53 7157 protein coding EJ -- Tumor Accelerate Cell morphological analysis//Flow cytometry//PI staining//BrdU assay//SA-β-gal activity assay "Proliferation of EJ–p53 cells overexpressing wt p53 (2tet) was almost completely inhibited.Tet removal led to obvious p53 nuclear staining by 24 h with the cells exhibiting increased size and a flattened morphology with elongated cellular processes, as well as enlarged nuclei.EJ–p53 cells exhibited greatly reduced BrdUrd incorporation within 24 h after the removal of tet with the fraction in S phase declining to ~5% by 2–3 days .Greater than 95% of such cells showed positive staining for SA-β-gal, which colocalized to cells possessing the senescent morphology ." -- -- -- -- -- -- -- -- Human L Others 9275177 Sen_G_1018 CDKN2A 1029 protein coding TIG-3 -- Aging Accelerate SA-β-gal activity assay "The both number of cells and [3H]thymidine incorporation are signi cantly decreased in H-ant-p16wt treated cells. Similar results were obtained using synchronized TIG-3 cells.The cells were then xed and stained for SA-β-gal, described to be expressed by senescent cells [27]. The enlarged growth-arrested cells gave a positive staining reaction." pRb -- Western blot "pRB is phosphorylated following serum stimulation in cells treated.However, pRB phosphorylation was not observed in cells treated with the H-ant-p16wt protein." -- -- -- -- Human L Others 9607312 Sen_G_1019 RAF1 5894 protein coding IMR-90 -- Aging Accelerate Cell counting//SA-β-gal activity assay "Activation of GFPDRaf-1:ER arrested the proliferation of IMR-90 cells.Activation of GFPDRaf-1[YY]:ER in IMR-90 cells elicited the expression of acidic β-galactosidase, which was apparent within 2 3 days and maximal by 5 6 days after addition of 4-HT." p16 Upregulation Western blot//SA-β-gal activity assay//BrdU assay//Flow cytometry "Following activation of GFPDRaf-1:ER, we observed induced expression of the CDK inhibitors p16Ink4a.However, the level of expression of p16Ink4a did not decrease after GFPDRaf-1:ER inactivation, which may reflect the long half-life of both p16Ink4a mRNA and protein.IMR-90 cells expressing p16Ink4a became positive for SA–β-galactosidase activity.FACs can analysis revealed a threefold reduction in S-phase cells compared with the control infected populations." MEK-MAPK Activation Western blot "As described above, activation of GFPDRaf-1[YY]:ER led to the phosphorylation and activation of the p42/p44 MAP kinases. In the presence of PD, however, phosphorylation of the MAP kinases was reduced to levels comparable to those found in uninduced IMR-90 cells." Human L Others 9765202 Sen_G_1020 MORF4 10934 protein coding A9+F4 -- Aging Accelerate SA-β-gal activity assay The MORF 4-transfected clones achieved between 19 and 35 PD before ceasing proliferation. The cells then morphologically resembled senescent cells and were positive for the senescence-associated β-galactosidase activity. -- -- -- -- -- -- -- -- Human HL Others 9891081 Sen_G_1021 E2F1 1869 protein coding "WI-38,NIH-3T3" -- Aging Accelerate SA-β-gal activity assay "Wild-type E2F1, when constitutively overexpressed, unexpectedly arrested cell proliferation within two population doublings (PD) after selection.wild-type and D423G proteins, but not the other E2F1 proteins, also induced .50% of the cells to assume a flat morphology, typical of senescent cells." p14//p53 Upregulation//Upregulation Semi-RT-PCR//Western blot "E2F1 proteins that had transactivation activity (wild type and D423G), but not the other E2F1 proteins, strongly induced p14ARF mRNA.p14ARF was also highly expressed by replicatively senescent cells.p14ARF and E2F1 did not arrest growth or induce SA-β-Gal staining in cells that overexpress Mdm2(Mdm2 functionally inactivates p53).Indeed, wild-type E2F1 and D423G proteins, as well as the p14ARF protein, strongly induced p53." -- -- -- -- Human L Others 10594030 Sen_G_1022 CDKN2B 1030 protein coding "U-251 MG,Tp483 MG,U373 MG,SW1783" -- Aging Accelerate Cell counting//Cell morphological analysis//SA-β-gal activity assay//Telomerase Assay "Both inhibitors(p15 ,p16) were equally effective in causing complete growth arrest during a 7-day period.When U251 MG cells infected with Ad5CMVp15 or Ad5CMVp16 were stained for the SA-β-gal marker 8 days p.i., the morphologically changed, enlarged, and flattened cells were strongly positive in contrast to mock-infected or to Ad5CMVlacZ infected cells .In contrast, telomerase activity was efficiently repressed in both Ad5CMVp15- and Ad5CMVp16- infected cells, reaching an undetectable level at day 6." -- -- -- -- Rb -- Cell morphological analysis//SA-β-gal activity assay U373 MG cells infected with Ad5CMVp15 or Ad5CMVp16 showed no morphological changes even after 7 days.The U373 MG cell line(absent pRB expression) did not become SA-β-gal-positive after infection by adenoviruses expressing p15 or p16. Human L Others 10939591 Sen_G_1023 ETS1 2113 protein coding HDF -- Aging Accelerate Western blot "In normal circumstances, p16INK4a(senescence marker) levels increase substantially in senescent HDFs,we found that the Ets2 levelsdeclined .It was clear that the loss of Ets2 from senescent cells was in part compensated for by a reciprocal increase in Ets1 expression." -- -- -- -- -- -- -- -- Human L Others 11234019 Sen_G_1024 ID1 3397 protein coding HDMEC -- Aging Prevent Cell counting//Cell morphological analysis "HDMECs of transduced with LZRS-empty vector (HDMEC+vector) became senescent at approximately PD17 and demonstrated the enlarged, flattened morphology seen in the untreated cells.The HDMEC+Id-1 cells demonstrated a significant increase in replicative capacity and continued to divide until PD38." -- -- -- -- Rb Downregulation Western blot "In contrast, HDMECs+Id-1 showed slightly lower, but sustained levels of both Rb forms until senescence, when levels of pRb and ppRb were barely detectable (not shown)." Human L Others 12177246 Sen_G_1025 MAP2K6 5608 protein coding U2OS -- Aging Accelerate Colony formation assay//BrdU assay//Flow cytometry//SA-β-gal activity assay//Luciferase reporter assay "Relative to cells transfected with empty vector alone or with MKK6KA, ＜6% of the MKK6EE-transfected cells formed colonies.MKK6EE expression leads to a near complete loss of BrdUrd-positive cells, consistent with a G1/G0cell cycle arrest.We observed that 40% of MKK6EE-expressing cells are strongly positive for SA-βgal, consistent with a senescent phenotype.At 6 days after transfection ,lipofuscin was markedly increased in fluorescence of MKK6EE-expressing cells compared with controls." p21 Upregulation Western blot "Trikingly,p21Cip1, a cdk inhibitora markediy increase." p38 Activation SA-β-gal activity assay//Luciferase reporter assay "As expected, expression of MKK6EE in U2OS cells increased the activity, whereas kinase-dead MKK6 slightly reduced basal activity .Cellular Senescence was not observed with MKK6KA cells or MKK6EE-expressing cells, which were treated with SB202190." Human HL Others 12208764 Sen_G_1026 PNPT1 87178 protein coding "HO-1,melanoma cell,WM278,MEWO" -- Aging Accelerate MTT assay//Telomerase assay "Infection with Ad.hPNPaseold-35resulted in significant growth inhibition in all of the cells.Infection with Ad.hPNPaseold-35at an m.o.i.of 100 pfu/cell,but not with Ad.vec,inhibited telomerase activity by almost 60% at day 4 after infection." c-Myc Downregulation Northern blot The expression of c-myc mRNA began decreasing 2 days after Ad.hPNPaseold-35infection but not in uninfected or Ad.vecinfected cells even at 4 days after infection. -- -- -- -- Human L Others 12721301 Sen_G_1027 MXD4 10608 protein coding "293,HeLa" -- Aging Accelerate Colony formation assay//Growth curve assay//SA-β-gal activity assay//Western blot "Growth was repressed by about 4- and 2.5-fold in 293- and HeLa-hMad4 infected cells, respectively. This repression of cell proliferation was accompanied by an increase in contact inhibition in a soft agar colony assay.In the case of hMad4-infected cells, following a small and very slow proliferative phase from day 1 to day 26 following replating, cells completely stopped dividing at which point they acquire the morphological features of replicative senescence such as flat and enlarged morphology.hMad4-infected cells did express s-β-galactosidase expression while empty-vector- and hMad4L-P16-infected cells had a very low expression level.Only hMad4- infected cells had a high expression level of Pai-1 protein(senescence marker)." Max//c-Myc Binding//Downregulation Co-IP//CAT assay "Anti-Max and antihMad4 are able to co-immunoprecipitate hMad4 and Max, respectively.When c-Myc and Max were co-transfected in 293 cells, there was a 10-fold increase in CAT activity over cells transfected with the reporter construct alone. When hMad4 was co-transfected with c-Myc and Max at increasing amount, there was a 4-fold decrease in CAT activity, similar to the activity seen with transfection of c-Myc alone." -- -- -- -- Human HL Others 12761891 Sen_G_1028 SOD1 6647 protein coding "WI-38,WI38-SV40" -- Aging Prevent Cell morphological analysis//SA-β-gal activity assay "In contrast,the SOD1 RNAi-transfected cells no longer divided.In addition the SOD1 RNAi morphology was aberrant and cells had an increased cell area and flattened appearance .WI38 SOD1 RNAi cells exhibited significantly stronger activity of senescence-associated β-galactosidase than the control cells." p53 -- Immunostaining "In contrast to control cells, SOD1 RNAi-transfected cells displayed the p53 protein in the nucleus and at elevated expression levels." -- -- -- -- Human L Others 12871978 Sen_G_1029 MAP2K1 5604 protein coding "HIEC,IEC-6" Small intestine Aging Accelerate Cell counting//Western blot//Cell morphological analysis//SA-β-gal activity assay "By contrast, caMEK-expressing HIEC cells did not proliferate and stopped accumulating well before reaching confluence.Ectopic expression of caMEK significantly inhibited thymidine kinase gene expression by 50%.Morphological examination of HIEC cells arrested by caMEK revealed that cells remained attached to the plates for at least 1 mo (data not shown) and acquired a large and flat morphology.Moreover, these HIEC cells accumulated SA-β-galactosidase." -- -- -- -- MEK-ERK -- Western blot Serum stimulation of HIEC and IEC-6 cells exhibited rapid and maximal activation of ERK1/2 within 10 min and persisted for up to 2 h. Nonstimulated caMEK expressing HIEC and IEC-6 cells showed a moderately increased basal level of ERK1/2 activities compared with pLXIN- and wtMEK-expressing cells. Human L Others 14701721 Sen_G_1030 NINJ1 4814 protein coding HuH-7 "HCC tissue,Liver" Hepatocellular carcinoma Accelerate Flow cytometry//SA-β-gal activity assay//Autofluorescence "Injurin1 overexpression increased the percentage of the total cell population in the G0–G1 phase, decreased the percentage of cells in the S phase. The percentage of SA-β-galactosidase-positive cells in ninjurin1-overexpressing populations was 26% in Nin1 and 21% in Nin2. In contrast, less than 1% of the parental and mock clones were SA-β-galactosidasepositive.Nin1 and Nin2 cells displayed intense cytoplasmic, granular fluorescence.Flow cytometric analysis revealed that both Nin1 and Nin2 cells showed a discernable increase in fluorescence in comparison to the mock clones." p21//Cyclin A//CDK2//CDK4//CDK6 Upregulation//Downregulation//Downregulation//Downregulation//Downregulation Western blot "There was no change in the level of p27, but the level of expression of p21 was greatly upregulated.The level of expression of cyclinA declined.The levels of expression of all CDKs(CDK2,CDK4,CDK6) were consistently suppressed by ninjurin1 overexpression." -- -- -- -- Human L Others 15464245 Sen_G_1031 SMURF2 64750 protein coding "BJ,WS1,WI-38" "Foreskin,Skin,Lung,Mammary Gland" Aging Accelerate Cell counting//SA-β-gal activity assay "Adventitious expression of Smurf2 to the level observed physiologically during replicative senescence resulted in arrest of proliferation of all three lines of early passage fibroblasts in a subconfluent state.During this acute transition, the early passage Smurf2-expressing cells acquired the property of positive staining for β-galactosidase activity at pH 6." -- -- -- -- p53//Rb --//-- Cell counting//Cell morphological analysis//SA-β-gal activity assay "The concurrent expression of both E6 and E7 prevent Smurf2 s effects as indicated by cell growth arrest in a subconfluent state, enlarged and flat cell morphology, and positive staining for -galactosidase activity at pH 6." Human HL Others 15574587 Sen_G_1032 DHCR24 1718 protein coding "REF,WI-38" -- Aging Accelerate Cell morphological analysis//BrdU assay//SA-β-gal activity assay "In contrast, upon Ras introduction, REF cells with Seladin-1 knockdown continued to proliferate, exhibited a condensed cell morphology, and were not stained in SA-β-gal assay .We observed that cells with Seladin-1 knockdown continued to show BrdU incorporation, cell proliferation and approximately fourfold less SA-β-gal staining after H2O2 treatment." p53//MDM2 Binding//-- Western blot "Silencing of Seladin-1 prevented Ras-induced p53 accumulation. We found that, first, Seladin-1 binds p53 and displaces Mdm2 from p53 in vitro.Second, recombinant Seladin-1 inhibits in vitro ubiquitination of Flag-p53 by Mdm2." Ras Upregulation Western blot Seladin-1 levels increased in response to Ras expression. Human HL Others 15577914 Sen_G_1033 LEO1 12319 protein coding 2BS -- Aging Accelerate MTT assay//Immunostaining//Flow cytometry//Cell morphological analysis//SA-β-gal activity assay "2BS/Leo cells showed complete growth inhibition similar to senescent cells (PD56).In contrast to the significantly increased G1 DNA content of 2BS/Leo, which was somewhat like senescent cells, RDL-Leo–reduced the G1 content of transfected cells.2BS/Leo cells showed increasing gross enlargement,flattening, and accumulation of granular cytoplasmic inclusions, like senescent cells (PD56).No SA-β-Gal activity was observed in 2BS/RDL-Leo–(PD48) and young (PD 27) cells, whereas almost all 2BS/Leo(PD 48) cells were strongly stained, as were senescent 2BS control cells (PD 56). In contrast, 2BS/Leo ceased cell division 7–12 PD (CPD 48-50) earlier than did normal cells." -- -- -- -- p16//p21//PTEN Upregulation//Upregulation//Upregulation Western blot "The results showed that introduction of Leo into 2BS cells affected all three pathways by increasing the expression of p16INK4a, p21WAF1, and PTEN." Human HL Others 15791002 Sen_G_1034 ING2 3622 protein coding MRC-5 -- Aging Accelerate Western blot//SA-β-gal activity assay//Colony formation assay//Crystal violet assay "ING2 protein level was four- to sixfold higher in senescent fibroblasts.ING2 overexpression induced premature senescence in PDL28 fibroblasts. The downregulation of ING2 resulted in a statistically significant increase only in the number of small colonies (containing 10 to 50 cells). Thus, suppression of ING2 expression results in an extension of the life span of human fibroblasts for a limited number of divisions." p53//p300 --//-- Immunostaining//Co-IP//Western blot "The colocalization of ING2 with p53 (Ser15) was dramatically enhanced in senescent fibroblasts.The complex formation between p53, ING2, and p300 was further corroborated in transient transfections in wild-type p53-containing U2OS cells, where immunoprecipitation of endogenous p300 also contained p53 and transfected Flag-ING2.With a constant amount of p300, increasing the amounts of purified ING2 protein led to a further two- to fourfold increase in the acetylation of lysine 382 on p53, as determined by the two detection methods. No p53 acetylation was detected with ING2 protein alone." -- -- -- -- Human L Others 16024799 Sen_G_1035 PRPF19 27339 protein coding "HUVEC,I38-neo-1 ,I38-SNEV-1" -- Aging Prevent SA-β-gal activity assay//Comet assay "I38-neo-1 cells entered growth arrest after around 8 PDs post-infection, where the cells showed the typical morphology of senescent cells and stained positive for SA-β-Gal . In contrast, I38-SNEV-1 cells at the same PD were characterized by a young morphology with no positive SA-β-Gal staining and entered replicative senescence around PD18pI.These data indicate that a high SNEV level correlates with a decrease of accumulated DNA damage during in vitro cultivation." -- -- -- -- -- -- -- -- Human L Others 16388800 Sen_G_1036 CSNK2A1 1457 protein coding IMR-90 Liver and Testis Aging Prevent SA-β-gal activity assay These reindicate that CKII activity decreases in IMR-90 cells during both replicative and H2O2-induced senescence.SA-β-gal staining in cells tended to be an increase in a dose-dependent manner by treatment with DRB.33 PDL of IMR-90 cells transfected with anti-CKIIa siRNA exhibited a higher rate of SA-β-gal staining comparedwith control cells. The increase rate in SA-β-gal staining from control was approximately 15-fold for CKIIa siRNA. p53//p21 --//-- Western blot "When the protein levels of p53 and p21Waf-1 were determined by immunoblot, expression of both proteins was upregulated in CKII inhibitor-treated cells, like in H2O2- induced senescent cells." -- -- -- -- Human L Others 16442104 Sen_G_1037 IRF3 3661 protein coding "BJ,IMR-90" -- Aging Accelerate Cell counting//SA-β-gal activity assay//Knockdown "overexpression of IRF3 in BJ and IMR90 cells was shown to decrease the rate of cell proliferation and increase the senescence cell population,as determined by senescence-associated β-galactosidase staining assay.When the cellular life span was determined by 3T6 cell culture protocol, IRF3-knockdown BJ cells were found to increase the cell proliferation rate and life span." p53 Activation Western blot "We found that p53 mRNA was not increased by IRF3, but p53 protein and its downstream target p21WAF1 were upregulated in IRF3-overexpressing BJ cells." -- -- -- -- Human L Others 16513254 Sen_G_1038 MYC 4609 protein coding "BJ,IMR-90" -- Aging Prevent Colony formation assay "We introduced into the c-myc+/- cells a retrovirus vector expressing human telomerase reverse transcriptase (hTERT) to immortalize them. Although hTERT clearly extended their lifespan, several attempts with different vectors failed to elicit long term immortalization, whereas the same vectors readily immortalized c-myc+/+ cells in parallel experiments." Bmi-1//p16 Binding//-- qRT-PCR//CHIP "As expected, retrovirus- mediated overexpression of c-Myc in normal HDFs resulted in Bmi-1 mRNA induction.we demonstrated direct binding of c-Myc protein to the E-box in the bmi-1 promoter by chromatin immunoprecipitation (ChIP) analysis.In the absence of ectopic Bmi-1, lentivirus vector-expressed c-Myc shRNA elicited a 2-fold up-regulation of p16 mRNA within 3 days of infection. Ectopic Bmi-1 expression alone resulted in repression of p16 mRNA levels, which remained low after c-Myc knockdown." -- -- -- -- Human L Others 16537449 Sen_G_1039 PIAS4 51588 protein coding WI-38 -- Aging Accelerate Growth curve assay//Cell morphological analysis//SA-β-gal activity assay//Immunostaining//Flow cytometry//SAHF "We found that cells constitutively expressing PIASy irreversibly arrested growth 7 10 days postselection, whereas other PIAS proteins had no effect available with this article online).PIASy-overexpressing cells also exhibited a flat cell morphology, and more than 80% of the cell population stained SA-β-gal positive.These assays clearly demonstrated that PIASy-expressing cells were completely devoid of proliferative potential and predominantly arrested in G1 .We also observed formation of SAHFs in PIASy-expressing cells." E6 -- Western blot "Addition of PIASy, but not of other PIASs, to the reaction strongly enhanced the formation of HDAC1-SUMO conjugates, an effect that was abrogated by the presence of E6 or its 45/47/49 mutant.11E6 was also unable to inhibit PIASy-induced senescence, whereas E6 mutant 45/47/ 49 still did." p53-p21//Rb-p16 Activation//Activation Western blot These data corroborate the increased p21 protein levels we observed in PIASy-transduced fibroblasts and suggest that sumoylation might contribute to PIASy-mediated activation of p53 in the context of PIASy-dependent senescence.PIASy corepressed the cyclin E promoter in an Rb- and dosedependent manner.Loss of PIASy using siRNA in SAOS-2 cells caused a significant derepression of the cyclin E promoter in the presence of Rb. Human L Others 16793547 Sen_G_1040 KL 9365 protein coding MRC-5 -- Aging Prevent Cell morphological analysis//SA-β-gal activity assay//Knockdown "Downregulation of Klotho expression by RNAi significantly shortened replicative lifespan and reduced the growth rate of MRC-5 cells compared with mock-infected cells.Similar to the phenotype of senescent cells, Klotho RNAi cells were flattened and enlarged .we found a 15-fold increase in β-Gal activity in Klotho RNAi (1)-transduced cells compared to mock control and a sixfold increase for Klotho RNAi (2)." -- -- -- -- Insulin-IGF-1//p53-p21 --//Downregulation Western blot//SA-β-gal activity assay "The level of phosphorylated IRS1 is higher in MRC-5 cells treated with Klotho RNAi, suggesting an upregulation of signaling at this early step in the pathway. the levels of phosphorylated AKT and phosphorylated FOXO3 (the inActivate cytoplasmic form) are higher.Relative to control cells Klotho RNAi infected cells have similar levels of p16 protein but elevated expression of p21.Confirming the ability of p53 attenuation to restore replicative potential of Klotho RNAi cells, staining for β-Gal shows approximately the same number of positive cells in the double knockdown compared to the vector control or p53 RNAi cells ." Human L Others 17014852 Sen_G_1041 CXCL1 2919 protein coding "NOF150,NOF151" -- Ovarian cancer Accelerate Cell morphological analysis//SA-β-gal activity assay//Western blot//Immunostaining "We found that the cells became enlarged andflattened, showing growth arrest and increased SA-β-gal activity after 10 days of treatmentas compared with cells grown in normal medium without Gro-1,cells cultured in serum-free medium, or cells treated with the same concentration of mouse IgG.Gro-1 treatment of NOF151 cells led to expression of HP1β in granular foci and marked increases in cytoplasmic p16INK4A levels." -- -- -- -- p53 -- Cell morphological analysis//SA-β-gal activity assay//Western blot "Treatment of parental NOF150 cells with Gro-1 led to strong blue staining on day 10, demonstrating activation of cellular senescence by Gro-1, but disruption of p53 suppressed or eliminated this effect.Fibroblasts immortalized by the introduction of p53siRNA and hTERT in particular completely lost the senescent response to Gro-1 .Disruption of p53 also markedly attenuated p16INK4Aexpression, particularly in the presence of hTERT." Human L Others 17060621 Sen_G_1042 RNASEL 6041 protein coding "MEF,WI-38,HDF,SV-WI38,Swiss 3T3,NIH-3T3" -- Aging Accelerate Immunostaining//Cell morphological analysis//SA-β-gal activity assay "We found that RNase-L-expressing cells did not display nuclear labeling, suggesting that ectopic expression of RNase-L inhibited proliferation. In contrast,the nuclei of cells with low,background staining for RNase-L expression were strongly labeled.RNase-L induction resulted that these cells exhibited an increased cytoplasmic :nuclear volume and vacuolization, and a flattened, multinucleate morphology.Counting of the stained cells revealed that 62% of RNase-L-transduced WI38 HDFs were β-gal positive as compared to only 18% in the vector control cells. Replicative senescence was accelerated by nearly three weeks in the RNase-L WI38 as compared to vector control cells." -- -- -- -- -- -- -- -- Human L Others 17130839 Sen_G_1043 PCGF2 7703 protein coding MRC-5 -- Aging Accelerate SA-β-gal activity assay Mel-18 overexpression led to inhibition of cellular proliferation and induction of premature senescence in MRC-5 fibroblasts as determined by SA-β-gal staining. Bmi-1//c-Myc Downregulation//Downregulation Western blot//Knockdown "Our results indicated that similar to MRC-5 fibroblasts, stable overexpression of Mel-18 results in Bmi-1 down-regulation in MCF10A and MCF7 cells.Mel-18 overexpression led to down-regulation of c-Myc in multiple cell types.Western blot analysis of cells expressing Mel-18 shRNAs showed significant down-regulation of Mel-18 and up-regulation of Bmi-1 in these multiple cell types.As expected, knockdown of Mel-18 also up-regulated c-Myc expression." -- -- -- -- Human L Others 17151361 Sen_G_1044 MAPKAPK5 8550 protein coding MEF Skin Aging Accelerate SA-β-gal activity assay//Western blot "PRAK+/+ primary mouse skin fibroblasts (MSFs) became growth arrested and accumulated SA-β-gal upon transduction of an activated ras allele,Ha-RasV12, indicating that ras triggered premature senescence.Similarly, PRAK deletion abolished ras-induced senescence in primary mouse embryonic fibroblasts (MEFs）." -- -- -- -- Ras//p38//p53-p21 --//--//Activation Western blot//Luciferase reporter assay "Ras-induced PRAK activation was also detected in primary .Ras-and MKK3/6E-induced PRAK kinase activity was greatly reduced in both BJ and MEF cells by a specific p38 inhibitor, SB203580.This indicates that PRAK is activated by p38 during ras-induced senescence.In both BJ and wild-type MEF cells containing this p53 reporter, luciferase activity was stimulated significantly by Ha-RasV12 or MKK3E,confirming the induction of p53 transcriptional activity in senescence. The regulation of p53 activity by PRAK during senescence was confirmed by the observation that ras-induced expression of p21WAF1, an endogenous p53 target previously implicated in rasinduced senescence, was abolished in PRAK-deficient BJ and MEF cells." Human L Others 17254968 Sen_G_1045 PEX19 5824 protein coding "U2OS,Human immortalized postcrisis cell,Fibroblast" Human tumor tissue and normal tissue Tumor Accelerate Cell counting Examination of 5AZA-dC response in Pex19p-overexpressing cells in comparison to the vector-transfected control cells revealed that the overexpression of Pex19p made the cells more sensitive to 5AZA-dC-induced senescence.Consistent with their stronger growth arrest. -- -- -- -- p16 Upregulation Western blot Pex19p-overexpressing cells showed an enhanced induction of p16INK4A expression in response to 5AZA-dC treatment. Human L Others 17389721 Sen_G_1046 HSPA9 3313 protein coding "U2OS,Human immortalized postcrisis cell,Fibroblast" Human tumor tissue and normal tissue Tumor Accelerate Cell counting//Western blot "Noticeably, mortalin-overexpressing cells were significantly more sensitive to 5AZA-dC, and showed a stronger induction level of p16INK4A." -- -- -- -- p53-p21 Activation Western blot "5AZA-dC-treated cells indeed showed upregulation of p53 and its downstream effector, p21WAF1, in addition to p16INK4a, and exhibited nuclear translocation of the p53 protein. Whereas only 10% 20% of control cells showed nuclear p53, 90% of the 5AZA-dC-treated cells showed strong p53 staining in the nucleus ." Human L Others 17389721 Sen_G_1047 MMP9 4318 protein coding Daoy medulloblastoma cell line "Tumor tissue,Brain" Medulloblastoma Accelerate MTT assay//PI staining//Flow cytometry//SA-β-gal activity assay "Ad-MMP-9 infection led to a dosedependent decrease in cell proliferation.FACS analysis for nuclear DNA content by propidium iodide staining showed that cell growth was arrested in the G0-G1 cell cycle phase.Senescence, as indicated by β-gal staining (blue color), increased in a dose-dependent manner with Ad-MMP-9 infection." p21CIP//p16 --//-- Western blot We observed a significant induction of p21 with Ad-MMP-9 infection.The expression of p16 increased in a dose-dependent manner as cells reached senescence. ERK-MAPK -- Western blot We found that both ERK and pERK levels were increased in Daoy cells infected with Ad-MMP-9 compared with the controls. Human L Others 17510426 Sen_G_1048 EHF 26298 protein coding WI-38 -- Aging Accelerate SA-β-gal activity assay "When cells were maintained by regular feeding of fresh media,ESE-3b-expressing cells stopped growing significantly earlier than the mock-treated cells did.ESE- 3b-expressing cells also showed a higher level of SA-β-gal-positive cells than the mock-infected cells." p16 Upregulation Western blot The amount of p16 but not that of p21 was increased in ESE-3b-expressing cells. -- -- -- -- Human HL Others 17627613 Sen_G_1049 WNT2 7472 protein coding "RPE,WI-38" -- Aging Prevent DAPI staining//SAHF//SA-β-gal activity assay//Knockdown "There was a good correlation between knockdown of Wnt2 and recruitment of HIRA to PML bodies, formation of SAHF judged by DAPI staining, and expression of SA β-gal.Strikingly, Wnt3aconditioned medium or purified recombinant Wnt3a delayed HIRA s localization to PML bodies and formation of SAHF in cells passaged toward senescence .Likewise, exogenous Wnt3a delayed onset of cell senescence caused by extended growth of WI38 cells in culture." -- -- -- -- -- -- -- -- Human L Others 17643369 Sen_G_1050 ITGB4 3691 protein coding HUVEC -- Aging Accelerate SA-β-gal activity assay The percentage of positive cells increased significantly during VEC aging.The percentage of SA-βgal positive cells in integrin β4 siRNA treatment group (43.84% 2.17%) was much lower than that in the control group (59.65% 2.74%). PC-PLC//p53 Downregulation//Upregulation PC-PLC activity assay//Western blot "As a result, Ca2+-dependent PC-PLC was nearly not changed, but the activity of Ca2+-independent PC-PLC decreased obviously during senescence.Down-regulation of integrin β4 depressed P53 and ROS levels and inhibited the activity of Ca2+-independent PC-PLC significantly." -- -- -- -- Human L Others 17964297 Sen_G_1051 HSPB2 3316 protein coding MCF 10A -- Aging Prevent Colony formation assay//Cell morphological analysis//SA-β-gal activity assay "Hsp27 overexpression led to a significant increase in survival after doxorubicin treatment. significantly fewer Hsp27-MCF10A cells showed expression of acidic β-galactosidase and senescence morphology after treatment with doxorubicin, compared with control MCF10A cells." -- -- -- -- p53-p21 Downregulation Western blot "We observed a suppression of known downstream targets of p53, survivin, and cdc2 in shHsp27 cells.In fact, direct measurement of the degradation rates showed that the half-life of p53 in shHsp27 cells increased from 26to 42 min .Doxorubicin led to a pronounced accumulation of p21 in control cells but this increase was markedly suppressed in Hsp27-MCF10A cells.Nutlin-3 treatment for 48 h led to accumulation of p21 in control cells, but this accumulation was suppressed in Hsp27-overexpressing cells." Human L Others 18089808 Sen_G_1052 SPIN1 10927 protein coding HeLa -- Aging Accelerate PI staining//Flow cytometry//DAPI staining//CCK-8 assay//SA-β-gal activity assay "SPINDLIN1 over-expressing cells showed a heterogeneous DNA content, with broader G1 and G2 peaks and a substantial fraction of cells with 4N DNA content, at 29 days after transduction.Numerous large cells with multiple nuclei were easily visible under phase contrast microscope, and multinucleation was also clearly illustrated by DAPI staining .HeLa cell growth was delayed significantly by overexpression of SPINDLIN1.We indeed found that almost all adherent multinucleated cells were SA-βgal positive (3.2%( 1.4%) in SPINDLIN1 overexpressing cells verse 0.8% ( 0.3%) in mock transduction cells)." -- -- -- -- -- -- -- -- Human HL Others 18201843 Sen_G_1053 ID1 3397 protein coding MEF Embryo Aging Prevent Cell counting//Cell morphological analysis//SA-β-gal activity assay "3T9 protocol revealed premature growth inhibition of Id1-/- MEFs beginning at P-5.Morphologic evaluation of these cells demonstrated cytoplasmic enlargement and flattening of Id1-/- MEFs and 80–90% positive staining of these cells with senescence-associated β-gal(16) at P-8, indicating premature cellular senescence in contrast to normal (+/+) MEFs that were 10–20% positive." CDK2//CDK4//p16 --//--//Downregulation Western blot "Evaluation of cdk2 and cdk4 functions revealed 75% and 50% inhibition of kinase activity, respectively, in early passage MEFs lacking Id1.We analyzed human foreskin keratinocytes that constitutively expressed Id1 for p16 expression levels and found that p16 expression inversely correlated with Id1 expression in these cells. Furthermore, we detected no p16 expression by Western analysis in Id1-immortalized human foreskin keratinocytes." -- -- -- -- Human L Others 11427735 Sen_G_1054 NANOG 79923 protein coding HF-MSC Hair follicle Aging Prevent Cell counting//SA-β-gal activity assay "Cell growth assays showed that the ectopic expression of NANOG significantly increased the quantity of HF-MSCs by 1.4- and 1.2-fold,in comparison to cells in the vector group (control) on day 8.Ectopic NANOG expression significantly decreased the percentage of SA-β-gal-positive cells." p16//p21//p53//PBX1 Downregulation//Downregulation//Downregulation//Upregulation Western blot//Luciferase reporter assay "Western blotting showed that ectopic NANOG expression significantly upregulated PBX1 expression by 4.11±0.54-fold in comparison to that of the control group. Dual-luciferase reporter gene assays showed that ectopic NANOG expression significantly increased PBX1 promoter activity by 1.99±0.43-fold incomparison to that of the control group (P < 0.05). HNANOG decreased p16 expression by 0.38±0.18-fold (P < 0.01),p21 expression by 0.57±0.24-fold (P < 0.05) and p53 expression by 0.60±0.16-fold (P < 0.05)." Akt Activation Western blot "Western blotting showed that NANOG significantly increased PARP1 expression by 1.64 ± 0.09-fold (P < 0:01) and the expression of p-AKT/AKT by 3.14 ± 0.65-fold (P < 0:05),in comparison to the empty vector." Human L Others 31885790 Sen_G_1055 TXNIP 10628 protein coding MEF Kidney Aging Prevent Cell counting//Cell morphological analysis//SA-β-gal activity assay//Western blot//qRT-PCR "The growth rate of KO MEF cells was dramatically decreased, and the cells almost ceased to proliferate at late passages (P5).KO MEF cells were larger than WT MEF cells. KO MEF cells were stained with SA‐β‐gal and γ‐H2AX at Passage 5 (P5).The expression levels of g p53, p21, p16,PAI‐1, and PML are upregulated by western blotting and statistical analysis or quantitative real‐time PCR in KO MEF cells in comparision to WT MEF cells." -- -- -- -- Akt Downregulation Co-IP//Western blot "TXNIP was found to interact with AKT, which was also confirmed by immunoprecipitation assays in 293T cells.The expression of TXNIP inhibited the activity of AKT in a dose dependent manner in western blotting with phospho AKT antibodies and in vitro kinase assays." Human L Others 30168649 Sen_G_1056 AKT1 207 protein coding MEF Embryo Aging Accelerate Cell morphological analysis//SA-β-gal activity assay//BrdU assay "WT MEFs began senescing after passage, whereas Akt1/2 DKO MEFs began senescing after passage.This was also confirmed by the cells’enlarged and flattened cell morphology (data not shown),by SA-β-Gal staining,and by BrdU incorporation." ROS//FOXO//Sestrin 3 Upregulation//Downregulation//-- Immunostaining//qRT-PCR "We found that Akt1/2 DKO MEFs had significantly lower ROS levels compared with WT MEFs, whereas cells expressing activated Akt and Pten-/- cells had significantly higher levels of ROS.FoxO transcriptional activity was elevated in Akt1/2 DKO MEFs.We found that sestrin 3 (Sesn3) expression was highly induced by activated FoxO.Activated Akt induces ROS by increasing oxygen consumption combined with the inhibition of FoxO transcription factors." -- -- -- -- Human L Others 19061837 Sen_G_1057 EPHA3 2042 protein coding hTERT-RPE1 -- Aging Prevent SA-β-gal activity assay Our results showed that senescence was uniquely detected after knockdown of EPHA3 and not measurably apparent after knockdown of other EPHA RTKs expressed in hTERT-RPE1 cells . -- -- -- -- p16//p53 --//-- qRT-PCR//Immunostainings//Knockdown Characterization of the senescence signature showed that siRNA-mediated knockdown of EPHA3 leads to increased p16INK4a and p53 expression . Human HL Others 23324396 Sen_G_1058 MAP2K2 5605 protein coding IMR-90 -- Aging Accelerate Western blot//SA-β-gal activity assay "MEK induced p53 (about three-fold induction), p21 (aboutfive-fold induction), and p16 (about six-fold induction).Finally, MEK1Q56P-expressing cells exhibited high levels of SA-β-gal activity, with a percentage of positive cells similar to that observed in Ras-expressing or late passage IMR90 cells." -- -- -- -- -- -- -- -- Human L Others 9765203 Sen_G_1059 MAPK14 1432 protein coding "WI-38,MRC-5" -- Aging Accelerate Cell counting//BrdU assay//Cell morphological analysis//SA-β-gal activity assay "The growth rate of senescent cells was very low in the absence of SB203580.?However, in the presence of SB203580, a significant increase in growth rate was observed. More than 80% of both SB203580- treated and untreated young cells were labelled with BrdU. In contrast, only 18% of the SB203580-untreated senescent cells were BrdU-positive. SB203580 treatment also resulted in a dramatic decrease in the number of SA-β-gal-positive senescent cells and a morphological change from a flat cytoplasm to a spindleshaped one with a refractile appearance." -- -- -- -- -- -- -- -- Human L Others 12581156 Sen_G_1060 CDK4 1019 protein coding HDF -- Aging Prevent SA-β-gal activity assay//Cell counting//BrdU assay//Western blot "Forced expression of wtCDK4 generated colonies in which growth was rapid for ~4 weeks then began to slow and finally ceased after ~7 weeks in a state resembling M1. Similar growth kinetics were observed for wtCDK6 colonies with a final average size of 7*103 cells (range: 103 to 4*104;n=25) corresponding to 12.5 PD and for mCDK6 colonies with a final colony size of 8*103 cells (range: 0.6*103 to 2.7*104; n=14)corresponding to ~13 PD. Colonies expressing mCDK4 di?ered slightly in that growth started to slow after 2 to 3 weeks.Cells were large and attened with a granular cytoplasm and could be maintained in a proliferatively quiescent but viable state for prolonged periods of time with occasional refeeding. SAβ-gal activity was high (>95% of cells positive by histochemical assay), BrdU LI was very low (<2%) (data not shown), and TdT assay confirmed that apoptotic cells in all MintCDK populations were extremely rare.Western blot analysis confirmed that growth arrest was not due to loss of expression of the exogenous CDK genes." -- -- -- -- p53 Upregulation Western blot p53 was elevated (12 ± 37-fold) in all CDK overexpressors compared to the pBABEpuro control. Human L Others 12082615 Sen_G_1061 CDK6 1021 protein coding HDF -- Aging Prevent SA-β-gal activity assay//Cell counting//BrdU assay//Western blot "Forced expression of wtCDK4 generated colonies in which growth was rapid for ~4 weeks then began to slow and finally ceased after ~7 weeks in a state resembling M1. Similar growth kinetics were observed for wtCDK6 colonies with a final average size of 7*103 cells (range: 103 to 4*104;n=25) corresponding to 12.5 PD and for mCDK6 colonies with a final colony size of 8*103 cells (range: 0.6*103 to 2.7*104; n=14)corresponding to ~13 PD. Colonies expressing mCDK4 di?ered slightly in that growth started to slow after 2 to 3 weeks.Cells were large and attened with a granular cytoplasm and could be maintained in a proliferatively quiescent but viable state for prolonged periods of time with occasional refeeding. SAβ-gal activity was high (>95% of cells positive by histochemical assay), BrdU LI was very low (<2%) (data not shown), and TdT assay confirmed that apoptotic cells in all MintCDK populations were extremely rare. Western blot analysis confirmed that growth arrest was not due to loss of expression of the exogenous CDK genes." -- -- -- -- p53 Upregulation Western blot p53 was elevated (12 ± 37-fold) in all CDK overexpressors compared to the pBABEpuro control. Human L Others 12082615 Sen_G_1062 EWSR1 2130 protein coding "Lin-Sca1+c-Kit+,BM,HSPC" Blood Ewing sarcoma Prevent SA-β-gal activity assay//Knockdown//Flow cytometry "When we assessed senescence-associated β-galactosidase activity (SA-β-gal), Ews-/- embryos at a late stage of gestation had significantly higher levels of endogenous SA-β-gal activity compared with WT embryos, suggesting that senescence has already begun from prenatal stages.Cell-cycle analysis showed that a substantial number of Lin , Sca-1 , and c-Kit cells (81%) in Ews- /- mice progressed through the G1, S, and G2/M phases, which was in marked contrast with the Ews- /- mice (34% in G1, S, and G2/M)." -- -- -- -- -- -- -- -- Human L Others 21030557 Sen_G_1063 MAP3K7 6885 protein coding "IMR-90,MRC-5,GP293" -- Aging Accelerate qRT-PCR//Colony formation assay "A total of 33 kinases showed pro-senescence effects (particularly MAP3K7, PRKCD, and MATK)." -- -- -- -- NF-κB Activation Luciferase reporter assay "Kinases with strong pro-senescence effects, induced expression of SASP-component genes (IL1A, IL1B, IL6 and IL8) through activation of an NF-kappaB-dependent transcriptional program.The luciferase activity was strongly induced by the 3 kinases indicating that these prosenescent kinases activate the NF-κB pathway." Human HL Others 26583757 Sen_G_1064 PRKCD 5580 protein coding "IMR-90,MRC-5,GP293" -- Aging Accelerate qRT-PCR//Colony formation assay "A total of 33 kinases showed pro-senescence effects (particularly MAP3K7, PRKCD, and MATK)." -- -- -- -- NF-κB Activation Luciferase reporter assay "Kinases with strong pro-senescence effects, induced expression of SASP-component genes (IL1A, IL1B, IL6 and IL8) through activation of an NF-kappaB-dependent transcriptional program.The luciferase activity was strongly induced by the 3 kinases indicating that these prosenescent kinases activate the NF-κB pathway." Human HL Others 26583757 Sen_G_1065 MATK 4145 protein coding "IMR-90,MRC-5,GP293" -- Aging Accelerate qRT-PCR//Colony formation assay "A total of 33 kinases showed pro-senescence effects (particularly MAP3K7, PRKCD, and MATK)." -- -- -- -- NF-κB Activation Luciferase reporter assay "Kinases with strong pro-senescence effects, induced expression of SASP-component genes (IL1A, IL1B, IL6 and IL8) through activation of an NF-kappaB-dependent transcriptional program.The luciferase activity was strongly induced by the 3 kinases indicating that these prosenescent kinases activate the NF-κB pathway." Human HL Others 26583757 Sen_G_1066 FOXM1 2305 protein coding GBC-SD -- Gallbladder cancer Prevent SA-β-gal activity assay//Knockdown "Positive SA-β-gal staining was observed in approximately 20% of GBC-SD cells treated with FoxM1 siRNA, which was significantly higher than those in the control group and NC-shRNA group (P < 0.05)." -- -- -- -- -- -- -- -- Human L Others 25071344 Sen_G_1067 IGFBP3 3486 protein coding WI-38 -- Aging Accelerate SA-β-gal activity assay//Knockdown Incorporation of [3 H]thymidine was estimated between 48 and 72 h after the last stress.A decreased proliferative potential of HDFs (pb0.001) was observed in t-BHP- and ethanoltreated cells compared to control cells (no siRNA). The presence of IGFBP-3 siRNA strongly attenuated the reduction of the proliferative potential of HDFs observed after treatment with either t-BHP (pb0.01) or ethanol (pb0.001).S-A β-gal was monitored using microfluidic detection at 72 h after the last stress.The increased proportion of S-A β-gal-positive HDFs in t-BHP- or ethanol-induced premature senescence was largely attenuated in HDFs transfected with IGFBP-3 siRNA. TGF-β1 -- Immunofluorescence "We detected an increase of more than 20-fold in the relative transcript level of IGFBP-3 as well as an increase in IGFBP-3 protein level by immunofluorescence semiquantitative confocal microscopy. Moreover, neutralizing antibody against TGF-β1 added in the medium after the last EtOH- or t-BHP stress inducing premature senescence diminished the increased transcript and protein levels of IGFBP-3." -- -- -- -- Human HL Others 18329388 Sen_G_1068 RSL1D1 26156 protein coding "HEK293,2BS" -- Aging Prevent Western blot//Flow cytometry//Knockdown//SA-β-gal activity assay "p27Kip1 and PTEN protein levels were greatly decreased (4-fold for PTEN and 7-fold for p27Kip1) in cells with CSIG overexpression.As expected, silencing of CSIG in HEK 293 cells led to increased G0/G1 and reduced S compartments，cells overexpressing CSIG exhibited markedly elevated proliferation rates, showed increased S and reduced G1 compartments, and displayed a feature of young cells (lower SA-β-gal activity) compared with the empty-vector-transfected cells." -- -- -- -- PTEN-p27 Downregulation Knockdown//Western blot//qRT-PCR//RNA-binding protein (RBP) prediction analysis "Knockdown of PTEN in HEK 293 cells greatly diminished the effect of either overexpression or knockdown of CSIG on p27Kip1 expression.To our surprise, neither overexpression nor silencing of CSIG significantly affected the mRNA levels of PTEN although the PTEN protein level was decreased by 10-fold in CSIG overexpression and increased in CSIG silenced cells (4-fold for 20 nM siRNA and 10-fold for 50 nM siRNA) by Western blot analysis.By Western blotting of CSIG in the pulldown materials, PTEN 5 UTR and CR (but not 3 UTR) were targets of CSIG." Human HL Others 18678645 Sen_G_1069 MBP1 841633 protein coding HFF -- Aging Prevent Cell proliferation assay//Knockdown//SA-β-gal activity assay "HFF-MBPsi-4 exhibited a significantly slower rate of proliferation as compared with the HFF-control suggesting that depletion of MBP-1 inhibited cell proliferation. Detection of β-galactosidase activity at pH 6.0 is a known characteristic of senescent cells. After 48 h, cells were fixed and stained with X-gal for the detection of bgalactosidase activity. More than 70% of HFFMBPsi-4 displayed appearance of enlarged blue cells (panel a) in contrast to the control HFF cells, which failed to exhibit adetectable blue appearance." -- -- -- -- p53-p21 -- Knockdown//Western blot "We have observed p53 acetylation at Lys382 site and phosphorylation at Ser15 in MBP-1 knockdown HFF. These results suggest that MBP-1 knockdown in HFF results in activation of p53. Enhancement of p53 by phosphorylation and/or acetylation results in activation of its target genes. The cyclin dependent kinase inhibitor, p21 is one of the p53 target genes. Therefore, we examined whether an increase in p21 expression in HFF-MBPsi-4 was due to activation of p53 transcriptional activity. Our results demonstrated a significant increase in p21 in the HFF-MBPsi-4 as compared to HFF-control." Human HL Others 18852884 Sen_G_1070 IFNG 3458 protein coding "HUVEC,MEF" -- Atherosclerosis Accelerate Cell proliferation assay//Flow cytometry//SA-β-gal activity assay "Cell proliferation decreased slightly with IFN-g treatment in time- and dose-dependent manners. A decrease in cell proliferation by IFN-g treatment (more than 1000 U/ml) for 3 days was statistically significant (p < 0.05).The induction of cellular senescence by prolonged treatment with IFN-g was confirmed by an increase in SA-β-gal-positive cells in the treated cells compared to the control cell. IFN-γ treatment increased the G0/G1 cell population and decreased the S and G2/M cell population, suggesting that IFN-γ inhibited cell proliferation through G0/G1 arrest in the cell cycle. No difference in cell cycle status was observed in IFN-a treated cells for 6 days." -- -- -- -- p53 -- Knockdown//Western blot//SA-β-gal activity assay "Prolonged stimulation of IFN-g in the p16-knockdown (p16sh) cells increased SA-β-gal activity staining to a level similar to that in control cells. In contrast, no increase in SA-β-gal staining was observed in the p53-knockdown (p53sh) cells following prolonged stimulation with IFN-g. To obtain more evidence that IFN-g-induced senescence involves a p53 signaling pathway, we measured the effects of IFN-g on cellular senescence in wild, p16-/-, or p53-/- MEFs. We reproducibly noticed an increase in the number of SA-b-galpositive cells in p16-/- MEFs, but not in IFN-g-treated p53-/- MEFs, after prolonged stimulation with IFN-g. Therefore, these results suggest that IFN-g-induced cellular senescence is mediated through a p53-dependent pathway." Human L Others 19071156 Sen_G_1071 CPEB1 64506 protein coding "HFF,WI-38" -- Aging Prevent SA-β-gal activity assay//Knockdown//Western blot "After 68 population doublings, the mock-infected and shTETR-infected cells stopped dividing and assumed a flat senescent-like morphology. These cells also stained for β-galactosidase activity at acidic pH, a common marker for senescence. The shCPEB-infected cells, however, continued to grow, did not undergo a morphology change, and did not stain for β-galactosidase activity. Moreover, while mock-infected and shTETR-infected cells expressed high levels of p21CIP1 and p16INK4A, which is consistent with entry into senescence, the cells infected with shCPEB did not." p53 -- Western blot "Two days later, the cells were infected with a virus expressing CPEB; the cells were then analyzed for growth and p21, a target gene of p53.While CPEB induced senescence in cells lacking GSE-22, they were unable to do so if they contained the inhibitory peptide. Moreover, GSE-22 prevented p21 expression, thus demonstrating that it indeed inhibited p53 activity. These results indicate that CPEB-induced senescence requires p53 in human cells." -- -- -- -- Human L Others 19141477 Sen_G_1072 PLA2R1 22925 protein coding HDF -- Aging Accelerate SA-β-gal activity assay//Growth curve assay "We assessed the growth of control and PLA2R overexpressing WI38 cells. Cell growth blockade was observed in growth curve analysis and colony formation assay when PLA2R was overexpressed, and was mainly due to senescence induction, as PLA2R-overexpressing cells showed a strong SA-β-gal activity." -- -- -- -- p53 Activation Knockdown//Western blot "In shPLA2R-infected HDFs, p53, and its targets p21 and human double minute 2 (HDM2), decreased when compared with control cells. Phospho-Rb increased, suggesting that the cells were proliferating. Conversely, when PLA2R was ectopically expressed, the levels of p53, p21 and HDM2 increased when compared with control senescing cells and phospho-Rb was found to decrease." Human HL Others 19197340 Sen_G_1073 SUPT5H 6829 protein coding HeLa -- Aging Prevent Flow cytometry Tetracycline addition resulted in a gradual decrease in the populations corresponding to S and G2/M phases and a concomitant increase in the population corresponding to G1 phase. -- -- -- -- p53 -- Knockdown//Western blot We found that Spt5 depletion resulted in a significant elevation of the p53 protein level and a concomitant increase in the CDNK1A/p21 protein level. Human HL Others 19210550 Sen_G_1074 PRKCA 5578 protein coding "TIG-1,Human normal diploid cell" -- Aging Accelerate SA-β-gal activity assay//Growth curve assay "Adenoviral transduction of PKC-δ induced growth inhibition in TIG-1 cells. Bistratene A, a PKC-d activator, also strongly inhibited the growth of TIG-1 cells . Furthermore, PKC-δ transduction and activation caused enlarged and flattened cell morphology and augmented SA-β-Gal activity in TIG-1 cells, as was observed in the senescent TIG-1 cells." hTERT Downregulation qRT-PCR "We investigated whether PKC-δ when activated in senescent cells functions in repression of the hTERT gene. The results showed that hTERT repression in senescent TIG-1 cells was relieved following infection with PKC-δ-KN, demonstrating that PKC-δ functions in the repression of the hTERT gene in senescent cells." -- -- -- -- Human L Others 19279193 Sen_G_1075 TOP1 7150 protein coding "WI-38,IMR-90,U2OS,HDF" -- Aging Accelerate SA-β-gal activity assay//Knockdown//Colony formation assay//Growth curve assay "Colony formation by Top1-depleted HDFs correlated with a loss of senescence markers: the formation of senescence-associated heterochromatin foci and the appearance of senescence-associated β-galactosidase activity.Top1 knockdown extended the life span of WI38 cells to f10 more population doublings,Growth of U2OS or WI38 cells constitutively expressing GFPTop1 was much slower than that of control-transfected U2OS cells." -- -- -- -- p53 Activation Western blot "Furthermore, measurement of p53 activity in the presence of an increasing amount of Top1 with a constant amount of a p53 activity reporter revealed dose-dependent activation of p53 by Top1. To further confirm the involvement of the p53 pathway, we examined the effect of p53 inhibition by a dominant negative form of p53 (p53DN) over the proliferation arrest induced by GFPTop1. Interestingly, p53 pathway inhibition reverted efficiently the proliferation arrest induced by GFPTop1." Human HL Others 19435923 Sen_G_1076 RUNX1 861 protein coding Hs68 -- Aging Accelerate SA-β-gal activity assay We found that ectopic RUNX1 expression in these cells induced growth arrest with early onset and a senescence-like morphology with a flatter appearance than that seen with H-RASV12. p53//p38MAPK --//Activation SA-β-gal activity assay//Cell proliferation assay//Western blot "In p53-null MEFs, RUNX1-ETO did not induce senescence-like morphology or SA-β-gal staining (not shown), although a more marked growth delay was induced compared with that in RUNX1. These results suggest that both RUNX1 and RUNX1- ETO also have cell cycle inhibitory effects that are independent of p53, although it is clearly required for the full expression of the senescent phenotype. The abilities of RUNX1 and RUNX1-ETO to activate p38MAPK were examined using a specific antibody that recognizes two phosphorylation sites responsible for p38MAPK activation (Thr180 and Tyr182).Indeed, western blot analysis on day 7 of the culture period showed that both RUNX1 and RUNX1-ETO induced elevated levels of phospho-p38, with RUNX1-ETO being the more potent agonist, inducing phosphop38 to a similar degree as does H-RASV12.Notably, the levels of total p38MAPK remained unaffected." -- -- -- -- Human L Others 19448675 Sen_G_1077 PPM1D 8493 protein coding mesenchymal-stem-cell -- Aging Prevent XTT assay//Growth curve assay "Early-passage hMSCs (p5, open circle) and late-passage Wip1-hMSC (p12, filled square) proliferated normally, whereas late-passage Cont-hMSCs (p12, open square) underwent distinct cell growth arrest(To define ‘‘early-passage’’ and ‘‘late-passage’’ cells, we referred to the previous report that elucidated the growth kinetics of hMSCs [6]. According to those results, the growth of hMSCs is significantly retarded after passage number 10; thus, we used the term late passage for hMSCs grown more than 10 passages. This difference in cell growth rate was reproduced using the XTT assay: late-passage hMSCs (p13) barely proliferated after 1 week, whereas Wip1-hMSCs at the same passage level proliferated in a fashion similar to early-passage hMSCs.We therefore concluded that Wip1-hMSCs bypassed the cell growth arrest that occurs in late-passage hMSCs." p16//p38 Downregulation//Downregulation qRT-PCR//Western blot "Consistently, p16 mRNA levels determined by semiquantitative real-time PCR were upregulated in late-passage hMSCs, compared with early-passage hMSCs that had not experienced growth arrest. Interestingly, p16 mRNA levels in later passage Wip1-hMSCs were significantly lower than in the late-passage hMSCs.As expected, p38 activity determined by p38 in vitro kinase assay using ATF-2 as a substrate was increased by 50%, whereas p38 activity in Wip1-expressing hMSCs was decreased by 50%." -- -- -- -- Human L Others 19544416 Sen_G_1078 RPS6KA6 27330 protein coding "HCT116,IMR-90" Colon Colorectal cancer Accelerate SA-β-gal activity assay//Western blot//qRT-PCR "We tested RSK4 expression levels in IMR90 after inducing senescence by cumulative passages. Cells stopped proliferating, acquired the enlarged, flattened morphology typical of senescent cells, and were positive for SA-β-gal staining (data not shown). The senescent cells showed increased RSK4 expression on quantitative real-time PCR and Western blot ." -- -- -- -- -- -- -- -- Human L Others 19584160 Sen_G_1079 SIN3B 23309 protein coding MEF -- Aging Accelerate SA-β-gal activity assay//Knockdown "The percentage of Sin3B-/-MEFs that were positive for SA-β-gal staining was significantly lower than that of their wild-type counterparts at passage 6. This observation corroborates the noticeable differences in cell morphology after six to eight passages between Sin3B-/-and Sin3B+/+ MEFs, which appeared significantly more flat and enlarged, irrespective of the cell density." E2F -- CHIP "To showthe direct role of Sin3B in mediating E2F transcriptional control, we performed chromatin immunoprecipitation experiments. We found that Sin3B is strongly and specifically enriched at E2F target promoters upon RasV12 overexpression , but not at nonrelevant promoters such as oct4 , we showed that this enrichment for heterochromatin marks at E2F target promoters upon oncogenic stress requires the presence of Sin3B." p19-p53 -- Western blot//Knockdown "As previously reported, expression of p19ARF was induced upon transduction of activated Ras into wild-type cells . Surprisingly, in Sin3B / cells overexpressing activated Ras, the level of p19ARF protein was comparable with that detected in Sin3B+/+ cells. However, the amount of p19ARF detected in the absence of oncogenic stress was significantly higher in Sin3B -/-cells compared with Sin3B+/+ cells .Our observation that Sin3B-/- fibroblasts do not senesce upon RasV12 expression, in spite of high levels of p19ARF, strongly suggests that Sin3B functions downstream of p19ARF in oncogene-induced senescence. These results showthat, although Sin3B participates in the transcriptional repression of p19ARF under normal culture conditions, it is required for the induction of senescence upon activation of the p19ARF/p53 pathway." Human L Others 19654306 Sen_G_1080 PSMD14 10213 protein coding "H1299,HeLa,hTERT-HMEC1" -- Aging Prevent SA-β-gal activity assay//Knockdown//Flow cytometry The cell cycle was arrested at the G0-G1 phase in PSMD14 knockdown cells. A significant increase of SA-β-Gal positive cells was observed in PSMD14 knockdown cells (52%). CDK1//Cyclin B//CDC25C Downregulation//Downregulation//Downregulation Western blot "Three days post-transfection with PSMD14 siRNA,the levels of cyclin B1, CDK1 and CDC25C were decreased to 6%, 33% and 30% of the control level(NT siRNA knockdown sample), respectively (from 30%, 64% and 62% of NT siRNA control, respectively at 2 days of knockdown)." -- -- -- -- Human HL Others 19732767 Sen_G_1081 MECP2 4204 protein coding mononuclear cell Bone Aging Prevent Knockdown//Flow cytometry//SA-β-gal activity assay//Cell proliferation assay "We observed a significant reduction of S-phase cells, along with an increase of G1 cells in samples with partially silenced MECP2.The decrement of S-phase cells resulted in a decrease of cell proliferation as evaluated by cell count.We observed signs of senescence in cells treated with Ad-siRNA-MECP2, as detected by acid β-galactosidase, when compared with cells transduced with Ad-siRNACTRL." -- -- -- -- -- -- -- -- Human L Others 20065105 Sen_G_1082 NFE2L2 4780 protein coding HFL-1 -- Aging Prevent Knockdown//SA-β-gal activity assay//Cell proliferation assay//Western blot "Knockdown of Nrf2 resulted in inhibition of proliferation and induction of premature senescence in HFL-1 fibroblasts.The cells acquired the typical senescent morphology, exhibited a higher percentage of β-galactosidase positive staining and revealed increased levels of p16 protein,as compared with the HFL-1 cells transfected with scrambled siRNA." 18αGA -- qRT-PCR//Western blot//Immunoblotting "Specifically, treatment of HFL-1 young cells with 2 μg/ml of 18αGA for 2 h (optimal conditions, see next paragraph) induced both the RNA and protein expression levels of Nrf2. In support, we have observed reduced levels of Keap1 as well as increased levels of its modified isoform (a protein greater than 150 kDa). Moreover, cell fractionation experiments as well as immunofluorescence analysis under the same conditions revealed that treatment with 18αGA promoted Nrf2 translocation to the nucleus, a feature that concurs with its activation." -- -- -- -- Human L Others 20068043 Sen_G_1083 TRX1 824267 protein coding BJ -- Aging Prevent Knockdown//SA-β-gal activity assay As demonstrated by cessation of population doublings and by upregulated senescence-associated beta-galactosidase (SA-beta-gal) activity.Quantification of the SA-beta-gal staining showed that the percentage of cells with positive activity was significantly higher in cells infected with either of the shTRX1 constructs than for cells infected with the control shRNA construct against GFP. -- -- -- -- p53//p16 -- Western blot "TRX1 suppression in BJ cells led to persistent upregulation of p53/p21 and p16INK4a proteins .TRX1 suppression in the BJELT cells prevented the proliferation defect observed in normal BJ cells with intact p53 and p16INK4a pathways , verifying that either one or both pathways have functional roles in establishing the shTRX1-induced senescent phenotype as opposed to the proliferation defect arising from quiescence or another non-senescence-related mechanism." Human L Others 20074557 Sen_G_1084 CENPA 1058 protein coding "TIG-3,HeLa" -- Aging Prevent BrdU assay//Knockdown//Western blot//SA-β-gal activity assay "A BrdU incorporation assay (BrdU is a marker of DNA synthesis) demonstrated that CENP-A depletion in TIG3 cells reduced the proportion of cells transiting S phase of the cell cycle. The proportion of M-phase cells was also reduced in the CENP-A-depleted TIG3 cells, as determined by immunostaining for S10-phosphorylated histone H3 (a marker of late G2 and mitotic cells),immunoblotting analysis demonstrated that CENP-A depletion in TIG3 cells led to a reduction in the levels of cyclin B and CDC2, which are essential for cell cycle progression, and an elevation of the levels of the CDK inhibitors, p16INK4a and p21CIP1.TIG3 cells depleted of CENP-A also exhibited the increase in SA- β-Gal activity that is the cytological marker of senescent cells." -- -- -- -- p53 -- Knockdown//Proliferation assay P53 appeared to be essential for this proliferation arrest; CENP-A depletion in p53-depleted TIG3 cells did not stop proliferating. Human L Others 20160010 Sen_G_1085 ENDOG 2021 protein coding HUVEC Umbilical vein Aging Prevent Knockdown//SA-β-gal activity assay "We found that knocking down EndoG in both early passage and late passage cells led to a significant delay in cell proliferation.This was accompanied by an increase in the percentage of cells staining positive for senescence-associated ?-galactosidase, suggesting that depletion of EndoG from human endothelial cells induces premature senescence." -- -- -- -- -- -- -- -- Human L Others 20211237 Sen_G_1086 DDB2 1643 protein coding MEF Embryo Aging Accelerate Knockdown//SA-β-gal activity assay//Western blot "WT and DDB2 -/- MEFs at various passages were compared for the levels of p19Arf, which is critical for senescence in the MEFs. We consistently found a decrease in the levels of p19Arf mRNA in the DDB2 -/-MEFs.To measure oxidative stress-induced premature senescence, the cells were assayed for SA-β-Gal. Clearly, there was a significantly lower expression of SA-β-Gal in the DDB22-/-MEFs ." -- -- -- -- -- -- -- -- Human L Others 20351176 Sen_G_1087 snail 778762 protein coding "LNCaP,PC-3" -- Prostate cancer Prevent Knockdown//SA-β-gal activity assay//Cell activity assay "There was a significant 30–79% reduction in the relative ATP amount of SNAI1-siRNA-treated cells compared to cells treated with IR-siRNA after long-term knockdown of Snail for 5 days. In IRsiRNA-treated LNCaP cells, less than 5% stained positive for SA-β-gal, as compared to up to 30% of SNAI1-siRNA-treated LNCaP cells in some wells (p< 0.05）. As expected from the morphology, only a few PC-3 cells were positive for SA-β-gal,independent of siRNA treatment (data not shown)." -- -- -- -- -- -- -- -- Human L Others 20397042 Sen_G_1088 PTTG1 9232 protein coding "IMR-90,BJ-1,WI-38" -- Aging Accelerate SA-β-gal activity assay//Cell proliferation assay//Flow cytometry "The cell number of PTTG1- expressing cells did not increase after infection, indicating that PTTG1 inhibited proliferation of normal cells. Flow cytometry analysis revealed that the proportion of G1 cells was reduced in PTTG1-expressing cells.Our results clearly indicated that IMR90 cells expressing PTTG1 showed an induction of SA-β -gal activity." p53 Upregulation SA-β-gal activity assay//Cell proliferation assay//Knockdown "The level of PTTG1-activated p53 was decreased upon shRNA treatments. Significantly, the growth of PTTG1- expressing cells was increased upon knocking down p53 expression. The results indicated that reduced p53 function could restore PTTG1-induced growth inhibition in normal cells. The SA-β-gal activity was then checked to evaluate the effect of a lower p53 level on PTTG1-induced senescence. In the p53 knockdown cells, the number of SA-β-gal-positive cells was also significantly lower than that of the control cells." -- -- -- -- Human L Others 20452981 Sen_G_1089 Vdup1 38023 protein coding 2BS -- Aging Accelerate SA-β-gal activity assay//Western blot//Flow cytometry//Cell proliferation assay "The results showed that cells expressing VDUP1 arrested cell cycle progression at the G0/G1 phase,displayed the morphology of flat cells, stained positive for SA-β-gal, and formed SAHF 9 days after retroviral infection (5 days post-selection); moreover, molecular markers of senescence, such as increased p53, p21, p16, and decreased p-Rb levels, were also detected after VDUP1 overexpression." FOXO3a//miR-17-5p Binding//-- Luciferase reporter assay//qRT-PCR "Mutation of the FOXO-binding element decreased about 2.2- fold luciferase activity in senescent cells as compared with the wild-type construct. In contrast, the luciferase activity from the mutant construct slightly decreased as compared with the wildtype construct in young cells.To demonstrate that miR-17-5p interacts with specific target sequence localized in this region of VDUP1 3-UTR, an additional reporter construct was generated in which the 7-bp “seed” sequence (CGUGAAA) of two putative miR-17-5p target sites were mutated using PCR.The resulting construct, pVDUP1–3UTR/ miR-17- 5p, was transfected into young and senescent cells; this mutation dramatically increased luciferase activity in young cells, whereas only a slight elevation was observed in senescent cells." -- -- -- -- Human L Others 20656682 Sen_G_1090 TNFSF15 9966 protein coding CEP Peripheral blood Aging Accelerate Knockdown//SA-β-gal activity assay "The efficiency of the two knockdown constructs to downregulate endogenous TL1A versus control. TL1A knockdown cells were stained for SA-β-gal 7 and 9 days after transduction, and the amount of stained cells was quantified.For both TL1A-specific shRNAs, the amount of SA-β-gal–positive cells was decreased compared with control." -- -- -- -- -- -- -- -- Human HL Others 20675618 Sen_G_1091 BRG1 834543 protein coding MSC Bone marrow Aging Prevent Knockdown//SA-β-gal activity assay//Flow cytometry "BRG1 silencing reduced the percentage of S-phase cells and induced a decrease in apoptosis,We observed a small increase of G1 in MSCs with silenced BRG1 compared with controls (67.61 versus 58.33%). It is noteworthy that MSCs with silenced BRG1 had a significant lower percentage of S-phase cells (Po0.05; 29.11 versus 40.23%) and an increase of G2/M cells (3.28 versus 1.43%).We observed signs of senescence in cells treated with Ad-siRNA-2405, as detected by acid-β-galactosidase, compared with cells transduced with control Ad-siRNA." -- -- -- -- Rb-p53 -- Western blot//RT-PCR "In MSCs transduced with Ad-siRNA2405 or with control virus, we inhibited P53 with Ad-CMVE1A(YH47-928) and RB family proteins with AdCMV-E1A(RG2). Both RB and P53 seemed to have a role in BRG1-mediated senescence. In situ acid-bgalactosidase staining showed that in cells with silenced BRG1 the inhibition of P53 and of RB reduced the percentage of senescent cells . In contrast, protection from apoptosis seemed to rely mainly upon the RB family, because inactivation of these proteins significantly increased the percentage of apoptotic cells, reaching a percentage higher than that observed when BRG1 was silenced." Human L Others 20697355 Sen_G_1092 CHK1 852577 protein coding HCT116 -- Colorectal cancer Accelerate Knockdown//Flow cytometry//Immunofluorescence//SA-β-gal activity assay "A reduction in the cell numbers in the G1 or G2/M phases of HCT116 wt or p53–/–at 72 hrs following Chk1 siRNA transfection caused enhanced cell death reflected by increased Pre-G1 cell populations.cyclin B1 was found in the nucleus in 90% of p53–/–cells, allowing for the conclusion that p53–/– cells accumulated in late G2 just before mitosis .We found that wt cells establish a G1 arrest following Chk1-involved G2 arrest, which was associated with a senescent phenotype as shown by staining for β-galactosidase activity.In addition, wt cells showed the characteristic flattened and enlarged morphology in the majority of cells together with prolonged expression of p53 and p21WAF1." -- -- -- -- -- -- -- -- Human L Others 20716119 Sen_G_1093 NEK6 10783 protein coding "IMR-90,EJ" -- Aging Prevent SA-β-gal activity assay "While EJ-vector control cells became flattened and enlarged after p53 expression, EJ-Nek6 cells maintained normal morphology and grew continuously.In addition, whereas SA β-gal activity was significantly increased in EJ control cells starting 4 days after p53 expression, and a majority of EJ control cells showed SA β-gal activity at 6 day later, SA β-gal activity was significantly reduced in EJ-Nek6 cells indicating that the onset of p53- induced senescence was suppressed by Nek6 overexpression." -- -- -- -- -- -- -- -- Human L Others 21099361 Sen_G_1094 VENTX 27287 protein coding "293,IMR-90" -- Aging Accelerate SA-β-gal activity assay//Cell proliferation assay "Consistent with its role as an inhibitor of cell proliferation, induction of VentX expression is associated with significant inhibition of U2OS/VentXTet cell growth. Interestingly, VentX expression caused a striking morphological change with U2OS/VentXTet cells appeared to be enlarged and flattened. These cells displayed positive staining for SA β-galactosidase, a characteristic marker of cellular senescence." -- -- -- -- p53-p21//p16-Rb Activation//Activation Western blot//CHIP//Luciferase reporter assay//RT-PCR "We found that VentX expression led to a significant increase in the protein levels of p53 and two CDK inhibitors: p21 and p16ink4a ,Consistently, we showed that ectopic expression of VentX trans-activated the human p53 and p21 promoter-luciferase reporters in a dose-dependent manner.A ChIP assay therefore was performed to examine a potential direct interaction between VentX and the p53 promoter. The ChIP assay revealed a specific binding of VentX to the p53 promoter. The potential transcriptional activation of p16ink4a by VentX was suggested by the increased p16ink4a mRNA levels upon ectopic expression of VentX . Using p16ink4a promoterluciferase reporter assay, we further demonstrated that VentX promoted p16ink4a transactivation in a dose depend ent manner. Human p16 promoter also contains putative homeodomain binding sites.Consistently, the ChIP assay demonstrated a specific binding of VentX to the p16ink4a promoter." Human L Others 21325273 Sen_G_1095 YPEL3 83719 protein coding MCF-7 Mammary Gland Breast cancer Accelerate MTT assay//Knockdown//SA-β-gal activity assay "When compared to MCF-7 cells, shYPEL3-expressing MCF-7 cells showed a statistically significant increase in cell numbers when grown over 10 days. Using an MTT-based assay, YPEL3 knockdown for six days led to an increase in cell numbers while YPEL3 induction over the same period resulted in a decrease in cell number.MCF7 cells stably selected for the loss of YPEL3 grown in charcoalstripped serum did not demonstrate an elevation in cellular senescence." -- -- -- -- -- -- -- -- Human L Others 21671470 Sen_G_1096 IGFBP6 3489 protein coding HDF -- Aging Prevent BrdU assay//SA-β-gal activity assay//Growth curve assay "At the end of the lifespan, overexpression of IGFBP-6 enabled the cells to perform 4– 5 additional population doublings.This was accompanied by a significant increase in the cell proliferation rate as measured by BrdU incorporation and a significant decrease in apoptotic cell death determined by Annexin V-staining and propidium iodide staining at 90–95% of lifespan completed. 120 days after infection (ca. 95% of lifespan completed), nearly all the cells in the controls stained SA-β-gal positive, whereas roughly 50% of the IGFBP-6 expressing cells stained still SA-β-gal negative and displayed reduced p21Waf1/Cip1 protein expression relative to controls." -- -- -- -- -- -- -- -- Human L Others 21820463 Sen_G_1097 RUVBL2 10856 protein coding "A498,KRC/Y" The tumor specimens and corresponding tumor adjacent renal tissue Renal carcinoma Prevent Knockdown//SA-β-gal activity assay//Flow cytometry "The results showed that the percentage of β-Gal- positive in control RCC cells was significantly lower than that in reptin siRNA-treated cells,Flow cytometry analysis demonstrated that these reptin siRNA-treated cells underwent growth arrest at the G1 phase." -- -- -- -- -- -- -- -- Human L Others 22341977 Sen_G_1098 IGFBP7 3490 protein coding MCF-7 -- Breast cancer Accelerate SA-β-gal activity assay//Flow cytometry//Cell proliferation assay "Addition of CM from IGFBP-rP1-transfected cells to untransfected MCF-7 cells blocked cell proliferation and increased SA-β-gal activity, whereas CM from the control vector (pEGFP-N1)-transfected or untransfected cells failed to inhibit cellular proliferation and induce senescence.A significant increase in the cell population at the G0/G1 phase of the cell cycle was detected in IGFBP-rP1-transfected MCF-7 cells, which is one of the typical phenotypes in cellular senescence." p21 Upregulation SA-β-gal activity assay//Western blot//Cell proliferation assay The IGFBP-rP1-transfected MCF-7 cells had increased levels of p21 in comparison with the control cells.The results showed that cell proliferation block and increased SA-β-gal activity in response to IGFBP-rP1 were partially reversed by p21 knockdown. These results suggest that cellular senescence induced by IGFBP-rP1 is mediated at least in part by p21 up-regulation. -- -- -- -- Human L Others 22392074 Sen_G_1099 MCL1 4170 protein coding "HCT116,MCF 10A,MCF-7,MEL526,MEF" Tumor tissue Cancer Prevent SA-β-gal activity assay//BrdU assay//Colony formation assay "As control cells induced to senesce have significant increases in the number of nuclear PML bodies compared to untreated cells. However, cells overexpressing Mcl-1 had significant abrogation of senescent changes after treatment, including reduced SA-β-gal+ and no increase in PML foci compared to HCT116 empty vector cells. In contrast, doxorubicintreated control cells formed significantly fewer colonies compared to those overexpressing Mcl-1. In addition, using the BrdU incorporation assay, we observe that the proliferation of cells growing in drug-free media was equivalent regardless of the level of Mcl-1 expression and that doxorubicin treatment of control cells resulted in a marked decrease in BrdU incorporation." p53 -- SA-β-gal activity assay//Western blot We noticed that the accumulation of p53 in cells overexpressing Mcl-1 and treated with doxorubicin is the same as in control cells despite a significant difference in SAβ-gal-activity. -- -- -- -- Human HL Others 22451485 Sen_G_1100 TRPM8 79054 protein coding "BxPC-3,PANC-1" Pancreas/duct Pancreatic cancer Prevent Knockdown//SA-β-gal activity assay Results of the SA β-gal assay indicate that RNA interferencemediated silencing of TRPM8 induced senescence in the pancreatic cancer cell lines BxPC-3 and PANC-1. -- -- -- -- -- -- -- -- Human L Others 22555807 Sen_G_1101 POU5F1 5460 protein coding MSC -- Aging Accelerate SA-β-gal activity assay//Flow cytometry//Cell proliferation assay "When treated with doxycycline, OCT4A but not control vector-expressing MSC lost their spindleshaped structure and appeared fl at or shrunken in their morphology.There was a 50% decrease in the percentage of GFP-positive cells when OCT4A expression was induced, which was noticeable at 8 days post-doxycycline treatment, whereas no signifi cant changes were observed in the control vector-expressing cells.Cell-cycle analysis of the transgene-expressing cells showed that there was a G 1 cell cycle arrest during exogenous OCT4A expression in MSC." -- -- -- -- -- -- -- -- Human HL Others 22746537 Sen_G_1102 NEK4 6787 protein coding "HFF,BJ,293T,IMR-90,WI-38" Foreskin Aging Accelerate Knockdown//SA-β-gal activity assay//BrdU assay//RT-PCR "Long-term culture of BJ cells in which NEK4 expression is suppressed (BJ shN4) revealed an extension of the time to replicative senescence of 10 to 20 PD compared to BJ shGFP cells.At late passage (PD 55 to 60), BJ shN4 cells proliferated at nearly twice the rate of BJ shGFP cells .Suppression of NEK4 by shN4 no. 1 resulted in a decrease in p21 mRNA levels smaller than that observed upon expression of shN4 no. 2 (0.81- and 0.27-fold, respectively), but we note that the levels of p21 mRNA and protein were still reduced compared to those in BJ shGFP cells." -- -- -- -- -- -- -- -- Human HL Others 22851694 Sen_G_1103 ZMAT3 64393 protein coding MCF-7 Lung Aging Prevent Knockdown//SA-β-gal activity assay//Flow cytometry//Colony formation assay//Cell proliferation assay "MCF7 breast cancer cells, which were treated with an siRNA against Wig1 (Wig Si), exhibited decreased cell proliferation and clonogenic ability.Interestingly, Wig1 depletion induced typical senescence associated phenotypic changes: large and flattened morphology, positive senescence-associated β-galactosidase (SA-βGal) staining, and G1 cell-cycle arrest. No significant increase in the subG1 phase was observed." p21 Downregulation qRT-PCR//Western blot//Knockdown "Overexpression and depletion of Wig 1 downregulated and upregulated p21, respectively, at both the mRNA and protein levels.The qRT–PCR assay revealed that Wig1-wt and Wig1-mut1 reverted the p21 3' UTR reporter mRNA levels that were increased due to Wig1 depletion to levels similar to those of Con Si-transfected cells, whereas Flag–Wig1- mut2 failed to rescue these reporter mRNA levels." -- -- -- -- Human L Others 23085987 Sen_G_1104 PROX1 5629 protein coding "HepG2,Hep3B,Mahlavu,SK-HEP-1" Tumor tissue Hepatocellular carcinoma Accelerate BrdU assay//SA-β-gal activity assay//Western blot "We used SK-HEP-1 cells for overexpression study and found that Prox1 reduced proliferation (as indicated by BrdU incorporation) in a time-dependent manner with a 30% of inhibition was found at 72 h after Prox1 expression in these cells.we found that percentage of β-galactosidase-positive cells was significantly increased after Prox1 overexpression indicating an induction of senescence-like phenotype.we examined the expression of CDKI proteins in control and Prox1-overexpressing cells. Among these inhibitory proteins, only p53 was dramatically increased." p53 Upregulation Knockdown//SA-β-gal activity assay//Western blot We first demonstrated that increase of β-galactosidase-positive cells by Prox1 expression was totally abolished by knockdown of p53 by shRNA. Human telomerase reverse transcriptase (hTERT) and chemokine C-X-C motif ligand 1 (CXCL1) have been shown to be downregulated and upregulated separately by p53 during p53-mediated senescence. We found that Prox1 significantly repressed hTERT expression and this effect was abolished when p53 was inhibited by shRNA. -- -- -- -- Human HL Others 23291986 Sen_G_1105 SOX2 6657 protein coding HCT116 Tumor tissue Colorectal cancer Accelerate SA-β-gal activity assay//Flow cytometry//Cell proliferation assay//Western blot "Our cell proliferation and cell cycle analyses indicated that Sox2 infection into HCT116 cells suppressed proliferation by inducing accumulation of cells in G0/G1 and sub-G1 and reduction of S cell cycle phases compared with mock-infected cells. The attenuation of cell proliferation was associated with about 90% inhibition of anchorage-independent cancer growth in soft agar, a hallmark of cancer cell properties.We then examined senescence using an SA-?galactosidase assay and found that ectopic expression of Sox2 enhanced ?-galactosidase activity and protein level and suppressed Ki-67 protein level, a cell proliferation marker." ATG10 Upregulation Knockdown//Luciferase reporter assay//Western blot//RT-PCR//Immunochemical staining "Our cycledependent RT-PCR results demonstrated that the ATG10 mRNA level from Sox2-transfected cells was increased by about 2.5 fold compared with mock.Notably, Western blotting results and immunocytofluorescence against ATG10 indicated that ATG10 and LC3 protein levels were increased by overexpression of Sox2.We found that that deletion of putative Sox2 binding region significantly suppressed luciferase activity. Notably, ATG10 promoter activity was increased by overexpression of Sox2." -- -- -- -- Human HL Others 23451179 Sen_G_1106 YAP1 10413 protein coding IMR-90 -- Aging Prevent Knockdown//SA-β-gal activity assay//BrdU assay//Flow cytometry "Compared with the control, silencing YAP significantly induced cellular senescence. SA-β-gal staining and BrdUrd incorporation were used as readouts for senescence. Knockdown of YAP increases the expression of SA-β-gal and inhibits cell DNA replication.Furthermore, YAP downregulation also resulted in the inhibition of cell proliferation in a population doubling assay.As expected, YAP knockdown significantly reduced the S-phase and increased the G1-phase in the IMR90 cells." CDK6 -- qRT-PCR//Western blot//CHIP//Knockdown//SA-β-gal activity assay "In our experiments, YAP or TEAD knockdown significantly inhibited CDK6 mRNA level and protein expression in IMR90 cells.Further ChIP experiments confirmed the binding of YAP to these 2 sites, whereas we used the already identified YAP–TEAD target CTGF as a positive control. Furthermore, overexpression of CDK6 can partially rescue the increased senescence induced by YAP knockdown." TEAD//Rb-p53-p16 --//-- Knockdown//SA-β-gal activity assay//BrdU assay//qRT-PCR "We found that knockdown of TEAD1/3/4 also induces an almost identical cellular senescent phenotype as YAP silencing. Further solidifying our hypothesis that YAP regulates cellular senescence in a TEAD-dependent manner, we found that the knockdown of boIn our SA-β-gal staining experiments, silencing either p16 or p53 did not rescue the senescent phenotype caused by YAP knockdown th YAP and We also found that the individual knockdown of p53 or p16 could not rescue the YAP knockdown-induced senescent phenotype by conducting population doubling assay experiments. However, double knockdown of p53 and p16 partially rescued YAP knockdown-induced senescence , suggesting that YAP antagonized senescence through both the p53 and p16 pathways TEAD1/3/4 together did not lead to a further increased senescent phenotype, including SA-β-gal staining and BrdUrd incorporation as well as PDL." Human L Others 23576552 Sen_G_1107 NOTCH3 4854 protein coding BJ -- Aging Accelerate Knockdown//SA-β-gal activity assay//Western blot "Downregulation of Notch3 in mid-passage cells resulted in a delayed onset of replicative senescence and an extended replicative lifespan,Downregulation of Notch3 by shRNA resulted in decreased p21 expression, suggesting that Notch3 is required for elevated p21 expression in senescent cells." p21 Upregulation Knockdown//SA-β-gal activity assay//Western blot//Cell proliferation assay "Similar to BJ and WS1 fibroblasts, ectopic expression of NICD3 in LF1 cells induced senescence and p21 expression.However, the ability of Notch3 to induce senescence was significantly attenuated in p21- /- cells, as indicated by increased cell proliferation and decreased SA-β-gal staining compared with the parental LF1 cells. These results suggest that Notch3-induced senescence is partially mediated by p21." -- -- -- -- Human HL Others 23610446 Sen_G_1108 PEBP1 5037 protein coding "hTERT-immortalized p21?/?,LF1,PC-3,HCT116" -- Aging Accelerate Knockdown//SA-β-gal activity assay//MTT assay//Western blot "Elimination of RKIP could increase ERK activation and suppress the cellular senescence. Conversely, RKIP overexpression inhibited ERK activation and induced the cellular senescence (monitored by SA-β-Gal and DcR2 expression) in two kinds of p53-deficient cell lines.As we expected, overexpression of RKIP could suppress the cell proliferation, whereas RKIP knockdown could promote the cell proliferation." -- -- -- -- Ras-Raf-ERK Downregulation Knockdown//SA-β-gal activity assay//Western blot "Suppression of Ras-Raf-ERK pathway using Raf kinase inhibitor could induce the cellular senescence .Elimination of RKIP could increase ERK activation and suppress the cellular senescence. Conversely, RKIP overexpression inhibited ERK activation." Human L Others 23814485 Sen_G_1109 DPY30 84661 protein coding "IMR-90,NT2" -- Aging Prevent Knockdown//SA-β-gal activity assay//Cell proliferation assay//Western blot//Colony formation assay//Growth curve assay "The overall morphology of the DPY30 knockdown IMR90 cells changed, towards a more rounded and flattened phenotype with enlarged nuclei.SA-β-galactosidase assays showed that 490% of the shDPY30 IMR90 cells expressed Activate SA-β-galactosidase. Further, in proliferation assays, growth curves, and colony formation assays, shDPY30 IMR90 cells arrested and stopped proliferating." ID -- qRT-PCR//Western blot//Knockdown//SA-β-gal activity assay "In ID1 and ID3 overexpressing cells, DPY30 knockdown reduced mRNA levels, but not proteins levels, of ID1 and ID3, the shDPY30 cells with ID1 or ID3 overexpression regained their fibroblast features and began to proliferate again, albeit not to the same degree as the IMR90 control cells, while the shDPY30 cells remained in a senescence-like state ." -- -- -- -- Human HL Others 23872946 Sen_G_1110 ID4 3400 protein coding DU 145 -- Prostate cancer Accelerate SA-β-gal activity assay//Western blot//Immunostaining "Enlarged morphology and accumulation of cytoplasmic aggregates were observed in DU145+Id4 cells as compared to DU145 cells,indicative of senescence.An increase in senescence associated beta-galactosidase (SA-βgal) staining suggested that ectopic expression of Id4 in DU145 cells promotes senescence at a higher frequency than in un-transfected DU145 cells.Immunocytochemical analysis shown clearly indicate that Id4 up-regulates G1 cell- cycle regulators p21 and p2, as compared to DU145 cells. We also observed an increased p16 expression at protein and transcript levels in DU145+Id4 cells, compared to DU145 cells but its functional relevance in DU145 cells with respect to senescence remains obscure." E2F1 Downregulation Western blot "At 10 nm, a significant increase in senescence was not observed in DU145 cells. The basal (0 h) E2F1 expression was significantly lower in DU145+Id4 cells as compared to DU145 cells whereas E2F1 expression was almost undetectable in DU145+Id4 cells at subsequent time points." -- -- -- -- Human L Others 24122992 Sen_G_1111 RB1 5925 protein coding Primary human bone cell Bone Retinoblastoma Accelerate Knockdown//SA-β-gal activity assay//qRT-PCR "Consistent with a role in senescence, radiation-induced expression of Il1b, Il6, Il8/Mip2, and Mcp1 was markedly attenuated in Rb1fl/fl mice relative to that in wild-type mice . Confirming these findings, ex vivo studies using 4 Gy IR also showed decreased SA-β-Gal–positive staining (data not shown) and reduced protein expression of IL-6 and MCP-1 in calvaria from Rb1fl/fl mice compared with that in wild-type mice." -- -- -- -- -- -- -- -- Human HL Others 24231354 Sen_G_1112 HSPA5 3309 protein coding A2780 Bone Ovarian cancer Prevent Knockdown//SA-β-gal activity assay "Seven days after 3 μg/ml cisplatin treatment, <20% of the A23187-treated A2780 cells showed positive SA-β-gal staining, whereas cells treated with cisplatin only were almost all positively stained. To confirm the specific anti-senescence effect of GRP78, GRP78-siRNA was transfected into the A23187-treated A2780 cells. After 3 days, the treated cells were exposed to 3 μg/ml cisplatin and incubated with complete medium. After 6 days,the cells exhibited 90% positive SA-β-gal staining." p53//CDC2 --//-- Knockdown//Western blot "The results showed that GRP78 protein levels were significantly decreased after GRP78-siRNA transfection in the A2780 cells. Accompanied by knockdown of GRP78, P53 levels were significantly increased and CDC2 levels were significantly decreased. There were no changes in the protein levels of P21 and p-CDC2. This suggests that P53 and CDC2 are involved in cisplatin-induced senescence of A2780 cells." -- -- -- -- Human L Others 24756776 Sen_G_1113 ERRFI1 54206 protein coding WI-38 -- Retinoblastoma Accelerate SA-β-gal activity assay "Myc–Mig-6 overexpression increased cell senescence as evidenced by the elevated number of the SA-?-gal positive, blue coloured cells compared to vector control transfected fibroblasts." -- -- -- -- ERK//AKT Activation//Activation Western blot "The strong signal for phosphorylated EGFR (pEGFR Tyr1173) observed in early passage cells at 5 min of EGF stimulation indicated the quick and transient response . Late passage (P25) fibroblasts exhibited an attenuated acute response to EGF treatment as evidenced by the slightly decreased levels of Tyr1173 phosphorylated EGFR at 5 min.Downstream of the EGFR, ERK activity was sustained in late passage as compared to early passage cells, whereas AKT activity was massively down-regulated in late passage cells . Moreover, the expression level of p16INK4A, a typical senescence-associated protein, was generally elevated in late passage cells as compared to young fibroblasts." Human L Others 24815188 Sen_G_1114 LGALS3 404021 protein coding "AGS,MEF" Stomach Retinoblastoma Prevent Knockdown//SA-β-gal activity assay//Flow cytometry//Cell proliferation assay "Treatment of AGS cells with 10 mM galectin-3 siRNA (Gal3 siRNA) for 2 days reduced cell proliferation by 50%.The depletion of galectin-3 significantly increased the number of G1 cell cycle-arrested cells and the sub-G1 populations, whereas co-depletion of galectin-3 and p27KIP1 produced equivalent proliferation to scRNA control group cell populations.Galectin-3 -/- MEFs showed growth retardation when compared with galectin-3 +/+ MEFs.Depletion of galectin-3 resulted in growth retardation and premature senescence in the human foreskin fibroblasts." p27 -- Knockdown//Western blot//RT-PCR//SA-β-gal activity assay//Cell proliferation assay "Galectin-3 depletion was accompanied by decreased Skp2 and increased p27KIP1 expression. Although the expression of p16INK4A and p21WAF1/CIP1 remained unchanged, p14ARF levels were found to decrease slightly.Galectin-3 depletion reduced Skp2 mRNA but increased p27KIP1 protein expression without a change in the mRNA expression. This suggests that galectin-3 regulates the level of p27KIP1 protein by Skp2-mediated ubiquitin activity.Upon galectin-3 depletion, both the cell types exhibited growth retardation and additional p27KIP1 ablation reversed this growth retardation ." -- -- -- -- Human HL Others 24971481 Sen_G_1115 DHX9 1660 protein coding MRC-5 -- Aging Prevent Knockdown//SA-β-gal activity assay//Flow cytometry//Growth curve assay "DHX9-suppressed cells stained positive for SA β-gal, a distinguishing marker of senescent cells.DHX9 shRNA-expressing cells show a significantly higher percentage of SAβ -gal-positive cells compared with MRC-5 controls (50–57% for DHX9 knockdown cells compared with 3% for shFLuc.1309-expressing cells). Measurements of the growth rates of DHX9-depleted MRC-5 cells indicated these to be at least 2-fold lower than control cells and similar to hRAS V12-expressing cells . Cell cycle analyses showed an increase in the percentage of cells in the G0/G1 phase and a decrease in the percentage of cells in the S and G2 phases 14 days after infection of DHX9 shRNAs." -- -- -- -- p53 -- Knockdown//SA-β-gal activity assay//Western blot "Loss of p53 completely abolished the SA-β-gal staining and rescued the growth defect in DHX9 knockdown cells. In addition, loss of p53 eradicates the p21 and RB1 responses in the DHX9 knockdown cells, compared with the vector control .This demonstrates that p53 is essential for DHX9-induced senescence." Human HL Others 24990949 Sen_G_1116 HMGB1 3146 protein coding "A375,G361" Melanoma Melanoma Prevent Knockdown//SA-β-gal activity assay//Flow cytometry//Cell proliferation assay "Knockdown of HMGB1 expression resulted in a marked reduction of cell proliferation and importantly, this effect appeared to be nicely correlated with the level of HMGB1 expression.Relative to the control cells, HMGB1- deficient cells showed a significant delay in cell cycle progression.We noticed that the HMGB1 stable knockdown cells appeared to be flatter in shape and larger in size than the matched control cells.This morphology change prompted us to ask whether HMGB1 depletion might induce cellular senescence. Measurement of senescent-associated β-galactosidase activity showed a marked increase of β-Gal-positive cells." p21 -- Knockdown//SA-β-gal activity assay//Flow cytometry "The result indicates that depletion of p21 expression almost completely abrogated G1 cell cycle arrest induced by HMGB1 deficiency.In addition,knockdown of p21 expression also abolished the senescent phenotype in HMGB1 deficient cells." SP1 -- Luciferase reporter assay "Consistent with a Sp1-dependent and p53-independent mechanism of p21 regulation, mutation of Sp1 but not p53 binding sites abrogated p21 induction by HMGB1-deficience. Almost identical results were obtained in both A375 and SK-MEL-28 cell lines. These data together confirm a Sp1-depdent mechanism of p21 induction by HMGB1-depletion." Human HL Others 25051367 Sen_G_1117 SELENOH 280636 protein coding MRC-5 -- Aging Prevent Knockdown//SA-β-gal activity assay//Cell proliferation assay "Moreover, the complete growth inhibition of SelH shRNA MRC5 cells maintained under 20% O2 for 5 weeks could be partially rescued when grown in a 3% O2 incubator, although SelH shRNA MRC-5 cells remained to proliferate poorly (~300-fold slower) as compared to scrambled shRNA MRC-5 cells.the percent SA-β-gal positive cells 5 days after recovery were 17% and 70% in scrambled shRNA and SelH shRNA MRC-5 cells, respectively, in a 20% O2 incubator. When maintained under 3% O2, they dropped (p < 0.05) to 2% and 34 % in scrambled and SelH shRNA MRC-5 cells, respectively." -- -- -- -- ATM-p53 -- Knockdown//SA-β-gal activity assay//Proliferation assay "Treatment with Ku 60019 (5 μM), a specific ATM kinase inhibitor (53), for 28 days alleviated the slow proliferation phenotype by 3- fold in SelH shRNA MRC-5 cells, but slightly suppressed the proliferation in scrambled shRNA MRC-5 cells.Analyses of SA-βgal staining confirmed that the ATM kinase is required to senesce SelH shRNA cells (40% vs 5%) after being cultured for 28 days. SelH shRNA MRC-5 cells significantly accumulated (p < 0.05) additional H2AX and pATM Ser-1981 after 28 days in a 20% O2 incubator, but such induction was completely." Human L Others 25336634 Sen_G_1118 PRKCH 5583 protein coding MCF-7 -- Aging Accelerate Knockdown//SA-β-gal activity assay//Cell proliferation assay//Colony formation assay//Western blot "In response to H2O2 or etoposide, PKCη-knockdown cells showed significantly and reproducibly lower number of SA-β-galactosidase-stained cells compared with scrambled control cells.A lower number of proliferating cells were regrown out of senescent cultures of scrambled (sh scr 5-3) control cells compared with PKCη-knockdown (sh 3-3 and sh 2-2) cells in colony formation assays. Viability assays (Neutral Red) with additional PKCη-knockdown clones (sh 4-2 and sh 3-5) showed similar results (data not shown).p21Cip1 is required to induce senescence. Our results showed increased expression of p21Cip1 and p27Kip1 in PKCηexpressing cells (sh scr 5-3), which was markedly reduced in PKCη-knockdown cells upon etoposide treatment." -- -- -- -- -- -- -- -- Human L Others 25412309 Sen_G_1119 PDCD10 11235 protein coding IMR-90 -- Aging Accelerate Knockdown//SA-β-gal activity assay//qRT-PCR//BrdU assay "When CCM3 was inhibited in these cells,4-OHT could still increase the number of γH2AX-positive cells,showing that H-ras was Activate in both control and CCM3-deficient cells. However, it did not inhibit DNA synthesis, nor did it induce a senescent morphology or senescence-associated β-galactosidase activity." -- -- -- -- -- -- -- -- Human HL Others 25655101 Sen_G_1120 P3H1 64175 protein coding IMR-90 -- Aging Prevent Knockdown//SA-β-gal activity assay//Colony formation assay//Cell proliferation assay "The down-regulation of LEPRE1, LIMA1, MAGOHA, and MAGOHB caused a significant reduction in cell growth (p ≤ 0.05) at 4 days after transfection, when compared with a negative siRNA control. In parallel, we tested if knockdown of the nine genes also induces the SA-β-gal activity.SiRNAmediated down-regulation of LEPRE1, LIMA1, and MAGOHA and MAGOHB robustly increased the number of SA-β-gal-positive cells, compared to cells transfected with siRNA control.a clonogenic test performed on young IMR90 cells after the inhibition of LEPRE1, LIMA1, MAGOHA or MAGOHB by the corresponding siRNAs demonstrated that LEPRE1 or LIMA1 down-regulation strongly inhibited the formation of colonies, compared with a negative siRNA control, while MAGOHA or MAGOHB down-regulation hampered treated cells from resuming proliferation." -- -- -- -- -- -- -- -- Human L Others 26206181 Sen_G_1121 LIMA1 51474 protein coding IMR-90 -- Aging Prevent Knockdown//SA-β-gal activity assay//Colony formation assay//Cell proliferation assay "The down-regulation of LEPRE1, LIMA1, MAGOHA, and MAGOHB caused a significant reduction in cell growth (p ≤ 0.05) at 4 days after transfection, when compared with a negative siRNA control. In parallel, we tested if knockdown of the nine genes also induces the SA-β-gal activity. SiRNAmediated down-regulation of LEPRE1, LIMA1, and MAGOHA and MAGOHB robustly increased the number of SA-β-gal-positive cells, compared to cells transfected with siRNA control.a clonogenic test performed on young IMR90 cells after the inhibition of LEPRE1, LIMA1, MAGOHA or MAGOHB by the corresponding siRNAs demonstrated that LEPRE1 or LIMA1 down-regulation strongly inhibited the formation of colonies, compared with a negative siRNA control, while MAGOHA or MAGOHB down-regulation hampered treated cells from resuming proliferation." -- -- -- -- -- -- -- -- Human L Others 26206181 Sen_G_1122 MAGOHA 4116 protein coding IMR-90 -- Aging Prevent Knockdown//SA-β-gal activity assay//Colony formation assay//Cell proliferation assay "The down-regulation of LEPRE1, LIMA1, MAGOHA, and MAGOHB caused a significant reduction in cell growth (p ≤ 0.05) at 4 days after transfection, when compared with a negative siRNA control. In parallel, we tested if knockdown of the nine genes also induces the SA-β-gal activity. SiRNAmediated down-regulation of LEPRE1, LIMA1, and MAGOHA and MAGOHB robustly increased the number of SA-β-gal-positive cells, compared to cells transfected with siRNA control.a clonogenic test performed on young IMR90 cells after the inhibition of LEPRE1, LIMA1, MAGOHA or MAGOHB by the corresponding siRNAs demonstrated that LEPRE1 or LIMA1 down-regulation strongly inhibited the formation of colonies, compared with a negative siRNA control, while MAGOHA or MAGOHB down-regulation hampered treated cells from resuming proliferation ." -- -- -- -- -- -- -- -- Human L Others 26206181 Sen_G_1123 MAGOHB 55110 protein coding IMR-90 -- Aging Prevent Knockdown//SA-β-gal activity assay//Colony formation assay//Cell proliferation assay "The down-regulation of LEPRE1, LIMA1, MAGOHA, and MAGOHB caused a significant reduction in cell growth (p ≤ 0.05) at 4 days after transfection, when compared with a negative siRNA control. In parallel, we tested if knockdown of the nine genes also induces the SA-β-gal activity. SiRNAmediated down-regulation of LEPRE1, LIMA1, and MAGOHA and MAGOHB robustly increased the number of SA-β-gal-positive cells, compared to cells transfected with siRNA control.a clonogenic test performed on young IMR90 cells after the inhibition of LEPRE1, LIMA1, MAGOHA or MAGOHB by the corresponding siRNAs demonstrated that LEPRE1 or LIMA1 down-regulation strongly inhibited the formation of colonies, compared with a negative siRNA control, while MAGOHA or MAGOHB down-regulation hampered treated cells from resuming proliferation." -- -- -- -- -- -- -- -- Human L Others 26206181 Sen_G_1124 DUSP16 80824 protein coding PLC-PRF-5 "Human HCC sample,Tumor tissue" Aging Prevent Knockdown//SA-β-gal activity assay//TUNEL assay//Flow cytometry//BrdU assay "TUNEL and Propidium Iodide (PI) staining, respectively. TUNEL-positive signals could be detected in DNase I or chemotherapeutic drug doxorubicin treated cells, but not in DUSP16-silenced cells ,By contrast, we found that upon DUSP16 knockdown, the PLC/PRF/5 cells were blocked in the G1 phase, suggesting that these cells were unable to initiate DNA replication and maintained a growth-arrested state. We also noticed that DUSP16-silenced cells exhibited a lower BrdU incorporation rate when compared with control cells.In accordance with the impaired cell cycle transition, the cell cycle-related proteins CDK1, CDK2, CDK4 and cyclin E were markedly down-regulated in DUSP16-silenced cells. SA-β-Gal activity is a widely used marker for measuring cellular senescence. Intriguingly, the percentage of SA-β-Gal-positive cells significantly increased upon DUSP16 silencing." -- -- -- -- p53//Rb --//-- Knockdown//SA-β-gal activity assay//Western blot//qRT-PCR "Upon DUSP16 knockdown, p53 downstream effectors were dramatically up-regulated .In addition, we found that upon DUSP16 knockdown, the increase of SA-β-Gal-positive cells and the decrease of the number of viable cells were efficiently attenuated by shRNA-mediated p53 silencing .Interestingly, in PLC/PRF/5 cells, we identified that decreased levels of CDKs upon DUSP16 silencing led to reduced phosphorylation of Rb , suggesting that the activation of Rb is involved in DUSP16 silencing-induced senescence in p53 mutant liver cancer cells .In addition, in the HepG2 cell line that carries wild-type p53, our results showed increased phosphorylation of p53, as well as, reduced phosphorylation of Rb upon DUSP16 silencing ,suggesting that both p53 and Rb are likely downstream effectors of DUSP16 silencing-induced senescence in p53 wild-type liver cancer cells." Human HL Others 26381291 Sen_G_1125 GATA4 2626 protein coding "IMR-90,BJ" Brain Aging Accelerate SA-β-gal activity assay//BrdU assay "Ectopic expression of GATA4 induced senescence in human foreskin fibroblasts and IMR-90 fibroblasts,as shown by increased senescence-associated β-galactosidase (SA-β-Gal) activity and decreased 5-bromo-2′-deoxyuridine (BrdU) incorporation." TRAF3IP2//IL1A --//-- Knockdown//Western blot//qRT-PCR "TRAF3IP2 depletion partially blocked GATA4 activation of NF-kB, as assessed by the expression of SASP genes.Furthermore, ectopic expression of TRAF3IP2 partially rescued the reduced SASP caused by GATA4 depletion in IR-induced senescent cells.Depletion of IL1A reduced GATA4 activation of NF-kB, as assessed by the expression of SASP genes. Furthermore, activation of SASP-associated genes was transmitted to cells lacking GATA4 induction by conditioned medium from GATA4-induced cells.Thus, GATA4 appears to act, at least in part, through TRAF3IP2 and IL1A to activate NF-kB in activating the SASP." NF-κB Activation Western blot "GATA4 expression triggered NF-kB activation, and GATA4 depletion inhibited NF-kB activation during senescence ." Human HL Others 26404840 Sen_G_1126 NANOG 79923 protein coding "NIH-3T3 MEF,Mouse Fibroblast cell" -- Aging Prevent SA-β-gal activity assay//Cell proliferation assay "Nanog-TAT caused a strongly increased proliferation, yielding about four-fold more cells within 10 days as compared to the control.Approximately 6% of MP-hADFs cultured under normal conditions for 3 passages stained positive for SA-β-gal. In contrast, no SA-β-gal activity was detectable in MPhADFs cultured in the presence of Nanog-TAT." p27 Downregulation RT-PCR//Western blot "After 5 h of Nanog-TAT treatment, the RNA expression level of p27KIP1 was slightly reduced, to ～90% compared to fibroblasts cultured in control medium. After 8 h of Nanog-TAT application, the expression of p27KIP1 was diminished to～70%. After 21 h of Nanog transduction, we detected only ～25% p27KIP1 transcript as compared to cells incubated with control medium.Quantification demonstrates that 5 h of Nanog transduction is sufficient to reduce the p27KIP1 protein level to about half compared to control; 24 h of incubation with Nanog-TAT yielded a further decline of p27KIP1 to ～40% compared to control." -- -- -- -- Human L Others 26795560 Sen_G_1127 HRAS 3265 protein coding MEF Embryo Lung cancer Accelerate Cell morphological analysis "In the case of overexpressed oncogenic H-Ras, cells were analyzed 6 days post-infection when, as expected, cells had a clear senescent morphology." -- -- -- -- -- -- -- -- Human L genomic instability 19421407 Sen_G_1128 TFDP1 7027 protein coding TIG-3 -- Aging Prevent SA-β-gal activity assay//SAHF//Knockdown "Furthermore, several features of cellular senescence, such as a substantial increase in senescence-associatedβ-galactosidase (SA-β-gal) activity and senescence-associated heterochromatic foci (SAHF) were observed in DP1 knock-down cells,but not in cells expressing flag-tagged DP1 that is resistant to RNAi (unpublished data)." -- -- -- -- -- -- -- -- Human L delay aging 15716376 Sen_G_1129 MIR200A 406983 ncRNA Keratinocyte -- Aging Accelerate qRT-PCR//Western blot "Altogether, these findings point miR-200a as key player in primary human keratinocyte aging since its overexpression reduces oxidative DNA repair activity and may induce cell cycle arrest via p16 up-regulation and fuels chronic inflammation via NLRP3 activation." OGG1-2a//NRLP3//IL-1β//Bmi-1//p16 Downregulation//Upregulation//Upregulation//Downregulation//Upregulation Luciferase reporter assay//qRT-PCR//Western blot "HEK 293 cells have been cotransfected with a construct containing the OGG1-2a 3′-UTR downstream of a luciferase open reading frame and either with miR-200a or a miR-scramble. Relative luciferase activity was significantly down-regulated (~29%) upon miR-200a overexpression.As expected, following miR-200a expression ,a significant decrease of OGG1-2a expression was observed in both cell types.Of note, miR-200a overexpression induced also a significant increase of NRLP3, caspase 1, and IL-1β expression mainly in fibroblasts.Following miR-200a expression, Bmi-1 expression significantly decreased at both mRNA and protein levels in HEK 293 cells and primary human fibroblasts. Simultaneously, a significant increase of the senescence mediator p16 was observed ." -- -- -- -- Human HL cellular senescence 29765508 Sen_G_1130 LINC00673 100499467 ncRNA "A549,IMR-90" -- Lung cancer Prevent Knockdown//Flow cytometry//SA-β-Gal activity assay//Cell morphological analysis "LINC00673 knockdown caused a change of cellular morphology, namely increased cell size and adaptation of a flat cell morphology in both A549 lung cancer cells and IMR-9 normal lung fibroblast cells.The reduction of LINC00673 levels in A549 cells was accompanied by a prominent increase of cells in G0/G1-phases and a concomitant decrease of cells in S-phase .We confirmed a strong increase of SA-β-Gal positive A549 and IMR-9 cells 4 days after LINC00673 knockdown." -- -- -- -- p53 -- SA-β-Gal activity assay SiPOOL-mediated depletion of p53 was sufficient to overcome senescence triggered by LINC00673 depletion in A549 cells. Human HL cellular senescence 30499379 Sen_G_1131 CircLARP4 -- ncRNA "MHCC97L,HCCLM3" -- Hepatocellular carcinoma Accelerate Knockdown//Flow cytometry//SA-β-Gal activity assay "We performed flow cytometry to analyze the cell cycle distribution.CircLARP4 overexpression caused G1/S cell cycle arrest in MHCC97L, whereas knockdown of circLAPR4 exhibited the opposite effects in HCCLM3 cells.To verify our hypothesis, we performed SA-β-gal staining to determine the cellular senescence. Data showed that the percentage of SA-β-gal-positive cells in circLARP4-overexpressed MHCC97L cells was significantly increased." -- -- -- -- miR-761-RUNX3-p53-p21 -- Knockdown//Western blot//FISH//RIP//Luciferase reporter assay//Immunohistochemistry "Western blotting showed that the expression levels of p53 and p21 were upregulated in circLARP4-overexpressed MHCC97L cells and downregulated in sh-circLARP4 HCCLM3 cells.MiR-761 expression was remarkably decreased in circLARP4-overexpressed MHCC97L cells, while miR-761 expression was elevated in sh-circLARP4 HCCLM3 cells . FISH analysis in HCC cells showed that circLARP4 was co-localized with miR-761 in cytoplasm. Given that high degree of AGO2 occupancy in circLARP4 has been implicated previously,16 we performed RIP for AGO2 in HCC cells and showed higher expression levels of circLARP4 and miR-761 in AGO2 pellet compared to those in the control group . Subsequent luciferase reporter assays demonstrated that miR-761 was a direct target of circLARP4." Human HL cellular senescence 30520539 Sen_G_1132 MIR181A1 406995 ncRNA "ULM,MM" -- Uterine leiomyomas Accelerate SA-β-gal activity assay "ULM and MM cells overexpressing the miRNAs and cultured as spheroids were fixed and stained for SA-β-galactosidase. Spheroid MM and ULM showed a global senescence pattern and a significant increase in cellular senescence was observed in both ULM and MM spheroids with overexpressing miR-29b, miR-181a, miR-182, and miR-200c compared to vector controls.miR-181a and miR-182 overexpression resulted in an intense and diffusive pattern of β-galactosidase staining in ULM." Akt3//CCND2 Downregulation//Downregulation Luciferase reporter assay//Western blot MiR-181a overexpression can reduce the luciferase activity when transfection of luciferase with AKT3 3 -UTR in myometrial cells Transienttransfection of miR-181a in MM and ULM cells consistently reduced AKT3 expression.MiR-182 overexpression consistently represses CCND2 expression in all MM and LM cells tested. -- -- -- -- Human HL cellular senescence 30097674 Sen_G_1133 MIR182 406958 ncRNA "ULM,MM" -- Uterine leiomyomas Accelerate SA-β-Gal activity assay "ULM and MM cells overexpressing the miRNAs and cultured as spheroids were fixed and stained for SA-β-galactosidase. Spheroid MM and ULM showed a global senescence pattern and a significant increase in cellular senescence was observed in both ULM and MM spheroids with overexpressing miR-29b, miR-181a, miR-182, and miR-200c compared to vector controls.miR-181a and miR-182 overexpression resulted in an intense and diffusive pattern of β-galactosidase staining in ULM." Akt3//CCND2 Downregulation//Downregulation Luciferase reporter assay//Western blot MiR-181a overexpression can reduce the luciferase activity when transfection of luciferase with AKT3 3 -UTR in myometrial cells Transienttransfection of miR-181a in MM and ULM cells consistently reduced AKT3 expression.MiR-182 overexpression consistently represses CCND2 expression in all MM and LM cells tested. -- -- -- -- Human HL cellular senescence 30097674 Sen_G_1134 MIR26B 407017 ncRNA Fibroblast -- Aging Accelerate RT-PCR "RT-PCR analysis confirmed a gradual increase in the expression levels of each SA-miRNA with increase in population doublings in fibroblasts.In addition,transient overexpression of each individual SA-miRNAs in fibroblasts also reduced cell proliferation and increased p16 levels." p16 -- Knockdown "Stable knockdown of p16(HMEC.p16shRNA) generated a relative increase in PcG member expression, particularly in the case ofCBX7. Concordantly, SA-miRNA levels were reduced.We examined the impact of the transient loss of p16 on PcG and SA-miRNA expression.Transient transfection of presenescent HMECs with a potent siRNA targeting p16 increased the expression of each PcG member and repressed SA-miRNA levels in HMEC to a great degree than the changes observed." -- -- -- -- Human HL cellular senescence 24217920 Sen_G_1135 MIR181A1 406995 ncRNA Fibroblast -- Aging Accelerate RT-PCR "RT-PCR analysis confirmed a gradual increase in the expression levels of each SA-miRNA with increase in population doublings in fibroblasts.In addition,transient overexpression of each individual SA-miRNAs in fibroblasts also reduced cell proliferation and increased p16 levels." p16 -- Knockdown "Stable knockdown of p16(HMEC.p16shRNA) generated a relative increase in PcG member expression, particularly in the case ofCBX7. Concordantly, SA-miRNA levels were reduced.We examined the impact of the transient loss of p16 on PcG and SA-miRNA expression.Transient transfection of presenescent HMECs with a potent siRNA targeting p16 increased the expression of each PcG member and repressed SA-miRNA levels in HMEC to a great degree than the changes observed." -- -- -- -- Human HL cellular senescence 24217920 Sen_G_1136 MIR210 406992 ncRNA Fibroblast -- Aging Accelerate RT-PCR "RT-PCR analysis confirmed a gradual increase in the expression levels of each SA-miRNA with increase in population doublings in fibroblasts.In addition,transient overexpression of each individual SA-miRNAs in fibroblasts also reduced cell proliferation and increased p16 levels." p16 -- Knockdown "Stable knockdown of p16(HMEC.p16shRNA) generated a relative increase in PcG member expression, particularly in the case ofCBX7. Concordantly, SA-miRNA levels were reduced.We examined the impact of the transient loss of p16 on PcG and SA-miRNA expression.Transient transfection of presenescent HMECs with a potent siRNA targeting p16 increased the expression of each PcG member and repressed SA-miRNA levels in HMEC to a great degree than the changes observed." -- -- -- -- Human HL cellular senescence 24217920 Sen_G_1137 MIR424 494336 ncRNA Fibroblast -- Aging Accelerate RT-PCR "RT-PCR analysis confirmed a gradual increase in the expression levels of each SA-miRNA with increase in population doublings in fibroblasts.In addition,transient overexpression of each individual SA-miRNAs in fibroblasts also reduced cell proliferation and increased p16 levels." p16 -- Knockdown "Stable knockdown of p16(HMEC.p16shRNA) generated a relative increase in PcG member expression, particularly in the case ofCBX7. Concordantly, SA-miRNA levels were reduced.We examined the impact of the transient loss of p16 on PcG and SA-miRNA expression.Transient transfection of presenescent HMECs with a potent siRNA targeting p16 increased the expression of each PcG member and repressed SA-miRNA levels in HMEC to a great degree than the changes observed." -- -- -- -- Human HL cellular senescence 24217920 Sen_G_1138 MIR194-1 406969 ncRNA HUVEC -- Aging Accelerate qRT-PCR//SA-β-gal activity assay//Western blot//DAPI staining//BrdU assay "The increase of miR-194 expression in replicative senescence was validated in human umbilical vein epithelial cells (HUVECs) as well. In order to further confirm the function of miR-194 on cellular senescence, we transfected MEFs with miR-194 inhibitor, Antagomir 194 or Antagomir NC. The results unveiled that miR-194 inhibition by Antagomir 194 transfection conferred attenuated senescence phenotype and enhanced proliferation of MEFs. Consistently, the data of SA-β-gal staining and BrdU assay showed that increased expression of miR-194 conferred NIH/3T3 cells with enhanced H2O2-induced senescence and diminished cell proliferation. In contrast, inhibition of miR-194 protected NIH/3T3 cells against H2O2 induced senescence." DNMT3A Downregulation Dual-Luciferase reporter assay//Western blot "We further constructed a luciferase reporter vector carrying mutant fragment of DNMT3A-3’UTR-2. The results showed that mutation of the miR-194 binding site in the DNMT3A 3’UTR (mut-DNMT3A-2) significantly reversed the Agomir 194-dependent reduction of luciferase reporter activity.Subsequently, we examined the DNMT3A mRNA and protein expression levels in MEFs after transfection with Agomir NC or Agomir 194. The data showed that Agomir 194 transfection significantly suppressed the expression of endogenous DNMT3A at both mRNA and protein levels." -- -- -- -- Human L cellular senescence 27981676 Sen_G_1139 MIR22 407004 ncRNA "MRC-5,Fibroblast" -- Breast cancer Accelerate qRT-PCR//Western blot//DAPI staining//BrdU assay//Cell proliferation assay//Flow cytometry "Quantitative (q) RT-PCR analysis confirmed that miR-22 expression was increased in senescent TIG-3 and other fibroblasts and even up-regulated by more than fivefold in senescent MRC-5 cells.miR-22 also increased SA-β-gal activity miR-22 overexpression induced cell cycle arrest at G1 phase, accompanied by the decrease in percentages of S phase. The inhibitory effect of miR-22 on cell cycle progression has also been recently reported in other cancer cells." SIRT1//SP1//CDK6 --// --// -- "MiRanda,TargetScan,and PicTar//Luciferase reporter assay//Western blot//SA-β-gal activity assay" "The mRNAs of SIRT1 and Sp1 contain putative binding sites for miR-22 in their 3-UTRs, and each site is broadly conservative among mammals. CDK6 mRNA contains three miR-22 binding sites in the 3'-UTR, whereas it is different in conservation for each site.In SiHa and MDA-D3 cells, miR-22 significantly reduced the luciferase activities of the full-length 3'-UTR or WT SIRT1, SP1, and CDK6 site 1 and site 3) reporters, compared with the negative cont miR. Western blot analysis showed that overexpression of miR-22 markedly down-regulated SIRT1, SP1, and CDK6 in SiHa and MDA-D3 cells.We forced SiHa or MDA-D3 cells to express SIRT1, Sp1, or CDK6 using plasmid constructs lacking 3-UTRs of these genes. Indeed, miR-22–induced cell growth repression and SA-β-gal activity were partially rescued by the introduction of SIRT1, CDK6, or Sp1 in either SiHa cells or MDA-D3 cells (not depicted), although it seemed weak for the effect of Sp1 overexpression on cell growth, which might be because of the indirect role of Sp1 in the pRb pathway of senescence." -- -- -- -- Human HL cellular senescence 21502362 Sen_G_1140 MIR24-1 407012 ncRNA HDF -- Aging Accelerate SA-β-gal activity assay Notably a significantly higher percentage of senescent cells was observed in mir-24 transfected cells. TOP1 Downregulation Western blot This initial finding could be experimentally corroborated by showing that mir-24 overexpression results in TOP1 protein downregulation. -- -- -- -- Human L cellular senescence 26748253 Sen_G_1141 TERC 7012 ncRNA 2BS -- Aging Accelerate SA-β-gal activity assay//Immunostaining "hTERC-53 overexpressing cells showed a significantly faster senescence rate. Full length hTERC overexpressing cells also showed a similar phenotype, even though to a lesser extent, possibly the result of hTERC-53 accumulation due to overexpression of the full length RNA. Expression of hTERC-53r led to minor downregulation of the expression levels of the senescence markers such as p-AMPK and p16, and removal of the inhibitory modification of MnSOD." -- -- -- -- -- -- -- -- Human HL cellular senescence 30788732 Sen_G_1142 MIR34A 407040 ncRNA "HepG2,SMMC-7721,HHCC,SK-Hep-1" -- Hepatocellular carcinoma Accelerate SA-β-gal activity assay//Cell morphological analysis "We observed that introduction of miR-34a into liver cancer cells caused senescence-like phenotypes, with positive staining for senescence-associated β-galactosidase (SA β-gal) and enlarged cellular size." c-Myc//FoxM1//p53 Upregulation//Upregulation//-- qRT-PCR//Western blot//Luciferase reporter assay "The data showed that among the hTERT activators examined, FoxM1 and c-Myc were the most down-regulated genes by miR-34a overexpression.By target prediction, it turned out that FoxM1 and c-Myc have predicted binding sites for miR-34a in their 3 UTRs. With the transfection of miR-34a duplex (miR-34a) in cancer cell lines, we found that miR-34a reduced the protein levels of c-Myc and FoxM1 significantly.Our results showed that miR-34a inhibited luciferase activity significantly, whereas no effect was observed when their respective target sites were mutated, suggesting that miR-34a directly targets c-Myc and FoxM1 via binding to the 3UTRs in liver cancer cells.The miR-34a expression positively correlated with the p53 level (P < 0.05), which was consistent with the NCI-60 data.Our expectation, the p53 protein expression significantly increased after treatment with H2 O2 or cisplatin in the wildtype p53 cells and the levels of miR-34a also increased accordingly.Further, when endogenous p53 expression was knocked down by a p53 siRNA in the four p53 wild-type liver cancer cells, miR-34a expression was also attenuated." FOXM1-c-Myc -- qRT-PCR//Western blot "The effect of c-Myc overexpression rescued the FoxM1-mediated inhibition of hTERT, as revealed by qRT-PCR and western blot, suggesting c-Myc as one of the most important downstream factors of FoxM1. As altered expression of miR-34a would contribute to the impaired telomerase activity, we discovered similar effects of miR-34a mimics and FoxM1 siRNA on the hTERT expression, with possible synergistic effect when transfected together, as revealed by qRT-PCR and western blot. With no surprise, similar effects were also observed for miR34a mimics and c-Myc siRNA." Human L cellular senescence 25686834 Sen_G_1143 MIR433 574034 ncRNA A2780 -- Ovarian cancer Accelerate SA-β-gal activity assay//Western blot "Firstly,to investigate if miR-433 overexpression could induce senescence the protein levels of p16, phosphorylated Rb (p-Rb), and p21 were anaylzed in the miR-433-stable A2780 cells. This analysis showed that p-Rb was decreased in A2780 cells stably transfected with miR-433. Notably, there was no reciprocal upregulation of p16 and p21 in these cells . The senescence-associated β-galactosidase activity in these cells was determined by Western blot and β-galactosidase staining analyses and revealed a significant upregulation of senescence-associated β-galactosidase activity in miR-433-stable cells compared to controls(P < 0.001)." CDK6 Downregulation Western blot "In our earlier bioinformatics analysis of potential miR-433 targets, CDK6 was predicted by five of the seven databases as a candidate miR-433 target gene. Therefore, we set out to establish if miR-433 could regulate the expression of CDK6. By analyzing protein expression in both the miR-433 stable A2780 cells and the clonal derivative of this cell line, we observed a decrease in CDK6 expression. Additionally, transient overexpression of miR-433 in HeLa cells also demonstrated downregulation of CDK6. Moreover, the transient transfection of PEO1 cells with anti-miR-433 to inhibit miR-433, resulted in a demonstrable upregulation of CDK6." -- -- -- -- Human L cellular senescence 25684390 Sen_G_1144 MIR186 406962 ncRNA HCT116 -- Colorectal cancer Accelerate SA-β-gal activity assay "To examine the role of miR-186, miR-216b, miR-337-3p, and miR-76 in cellular senescence, we knocked down CKIIa in HCT116 cells by co-treatment with the same miRNA mimics.To assess the effect of miRNA knockdown on senescence, after transfection for 2 days the transfectants were then stained for SA-β-gal activity. HCT116 cells transfected with the four mimics exhibited a higher rate of SA-β-gal staining compared with control cells ." CKII//p53//p21/WAF1 Downregulation//Upregulation//Upregulation CKII activity assay//Western blot "When CKII activity was assessed using [c32P]ATP and CKII peptide substrate, extract from the cells transfected with the four mimics contained lower than 40% CKII activity compared to control extract.Immunoblot data showed that the expression levels of p53 and p21Cip1/WAF1 were upregulated in HCT116 cells treated with the four miRNA mimics in comparison with control cells." -- -- -- -- Human HL cellular senescence 23137536 Sen_G_1145 MIR216B 100126319 ncRNA HCT116 -- Colorectal cancer Accelerate SA-β-gal activity assay "To examine the role of miR-186, miR-216b, miR-337-3p, and miR-76 in cellular senescence, we knocked down CKIIa in HCT116 cells by co-treatment with the same miRNA mimics.To assess the effect of miRNA knockdown on senescence, after transfection for 2 days the transfectants were then stained for SA-β-gal activity. HCT116 cells transfected with the four mimics exhibited a higher rate of SA-β-gal staining compared with control cells." CKII//p53//p21/WAF1 Downregulation//Upregulation//Upregulation CKII activity assay//Western blot "When CKII activity was assessed using [c32P]ATP and CKII peptide substrate, extract from the cells transfected with the four mimics contained lower than 40% CKII activity compared to control extract.Immunoblot data showed that the expression levels of p53 and p21Cip1/WAF1 were upregulated in HCT116 cells treated with the four miRNA mimics in comparison with control cells." -- -- -- -- Human HL cellular senescence 23137536 Sen_G_1146 MIR337 442905 ncRNA HCT116 -- Colorectal cancer Accelerate SA-β-gal activity assay//Knockdown "To examine the role of miR-186, miR-216b, miR-337-3p, and miR-76 in cellular senescence, we knocked down CKIIa in HCT116 cells by co-treatment with the same miRNA mimics.To assess the effect of miRNA knockdown on senescence, after transfection for 2 days the transfectants were then stained for SA-β-gal activity. HCT116 cells transfected with the four mimics exhibited a higher rate of SA-β-gal staining compared with control cells ." CKII//p53//p21/WAF1 Downregulation//Upregulation//Upregulation CKII activity assay//Western blot "When CKII activity was assessed using [c32P]ATP and CKII peptide substrate, extract from the cells transfected with the four mimics contained lower than 40% CKII activity compared to control extract.Immunoblot data showed that the expression levels of p53 and p21Cip1/WAF1 were upregulated in HCT116 cells treated with the four miRNA mimics in comparison with control cells ." -- -- -- -- Human HL cellular senescence 23137536 Sen_G_1147 MIR760 100126348 ncRNA HCT116 -- Colorectal cancer Accelerate SA-β-gal activity assay "To examine the role of miR-186, miR-216b, miR-337-3p, and miR-76 in cellular senescence, we knocked down CKIIa in HCT116 cells by co-treatment with the same miRNA mimics.To assess the effect of miRNA knockdown on senescence, after transfection for 2 days the transfectants were then stained for SA-β-gal activity. HCT116 cells transfected with the four mimics exhibited a higher rate of SA-β-gal staining compared with control cells." CKII//p53//p21/WAF1 Downregulation//Upregulation//Upregulation CKII activity assay//Western blot "When CKII activity was assessed using [c32P]ATP and CKII peptide substrate, extract from the cells transfected with the four mimics contained lower than 40% CKII activity compared to control extract.Immunoblot data showed that the expression levels of p53 and p21Cip1/WAF1 were upregulated in HCT116 cells treated with the four miRNA mimics in comparison with control cells." -- -- -- -- Human HL cellular senescence 23137536 Sen_G_1148 HOTAIR 100124700 ncRNA A2780_CR5 -- Ovarian cancer Accelerate SA-β-gal activity assay//PI staining//Flow cytometry//Knockdown "In addition, IL-6 secretion, an established marker for cell senescence was increased by CDDP in A2780 p overexpressing HOTAIR compared with vector-transfected cells .DsiRNA knockdown of HOTAIR in A2780_CR5 cells reduced the number of senescent cells.We observe a decrease in S1 and an increase in G2 phase 48 h post CDDP treatment in HOTAIR expressing cells and a reversal of this effect in A2780_CR5 cells, suggesting that a subpopulation of cells undergo HOTAIR-dependent cell senescence." -- -- -- -- NF-κB//CHK1-p53-p21 --//-- Western blot//SA-β-gal activity assay//MTT assay "In HOTAIR overexpressing A2780p cells, SA-β-Gal-positive cell numbers were increased by high vs low evels of CDDP , and NF-κB inhibitor Bay-11 reduced the number of senescent cells,ctopic expression of HOTAIR in NF-κB knockdown cells rescued (Po0.05) proliferation and increased clonogenic survival.In HOTAIR overexpressing vs vector control cells, decreased p-p53 levels were observed and the level of p-Chk1 was essentially unchanged , suggesting ATR dependent activation of p53 by HOTAIR. Activation of p53 and p21 by HOTAIR was observed only during high CDDP treatment, indicated by p53 phosphorylation and p21 expression ." Human HL cellular senescence 27041570 Sen_G_1149 MIR145 406937 ncRNA T24 -- Urothelial carcinoma Accelerate TUNEL assay//SA-β-gal activity assay "miR-145 overexpression significantly suppressed cell proliferation. We found senescence to be induced in overexpressing T24 cells, although the number of apoptotic cells was not significantly increased ." syndecan-1 Downregulation qRT-PCR Overexpression of miR-145 significantly suppressed syndecan-1 mRNA. -- -- -- -- Human L cellular senescence 26514209 Sen_G_1150 MIR23A 407010 ncRNA K562 -- Chronic myeloid leukemia Accelerate SA-β-gal activity assay Strongly positive β-gal staining was observed in K562 cells transfected with miR-23a precursors compared with controls and the difference was statistically significant. -- -- -- -- BCR-ABL-PI3K-Akt-MMP9 -- qRT-PCR//Western blot//Luciferase reporter assay "Transfection with miR-23a precursor decreased the BCR/ABL 3′-UTR reporter activity in K562 cells, which strongly indicated that BCR/ABL was a target for miR-23a.Finally, we assessed the effect of miR-23a expression on BCR/ABL expression. Transfection of K562 cells with miR-23a precursor resulted in lower expression of BCR/ABL after 48h. Concomitant with decreased BCR/ABL expression, we observed reduction of its downstream targets PI3K, Akt and MMP-9, as we previously reported8." Human HL cellular senescence 25213664 Sen_G_1151 MIR221 407006 ncRNA Fibroblast -- Aging Accelerate Flow cytometry//qPCR "The impact of miR-221/222 on cell proliferation was investigated by FACS analysis and showed that miR-221/222 promoted the accumulation of HDFs in G1/S cell cycle phase. We then analysed the expression of genes implicated in cell cycle progression in miR-221/222 HDFs. Transient expression of miR-221/222 in both HFL-1 PDL28 and MRC-5 PDL31 HDFs resulted in a reduction in the expression of E2F1 and Cdc6, and in the up-regulation of p21Cip1." -- -- -- -- -- -- -- -- Human HL cellular senescence 28658612 Sen_G_1152 MIR222 407007 ncRNA Fibroblast -- Aging Accelerate Flow cytometry//qPCR "The impact of miR-221/222 on cell proliferation was investigated by FACS analysis and showed that miR-221/222 promoted the accumulation of HDFs in G1/S cell cycle phase. We then analysed the expression of genes implicated in cell cycle progression in miR-221/222 HDFs. Transient expression of miR-221/222 in both HFL-1 PDL28 and MRC-5 PDL31 HDFs resulted in a reduction in the expression of E2F1 and Cdc6, and in the up-regulation of p21Cip1." -- -- -- -- -- -- -- -- Human HL cellular senescence 28658612 Sen_G_1153 H19 283120 ncRNA NPC -- Disc degenerative disease Accelerate SA-β-gal activity assay//Immunofluorescence "SA-β-Gal positive cells and Collagen I content were increased, while telomerase activity was decreased by H19 overexpression." miR-22 Downregulation Luciferase reporter assay//qPCR "In NPCs, H19 and miR-22 negatively regulated each other. The results showed that the luciferase activity of wild-type vectors, wt-19 and wtLEF1 3 UTR could be suppressed by miR-22 mimics while amplified by miR-22 inhibitor; after mutating the predicted miR-22 binding site, the changes of luciferase activity were totally eliminated." Wnt-β-catenin -- Spearman's rank correlation analysis "Furthermore, H19 expression was positively correlated Wnt3 and β-catenin expression in tissue specimens, respectively.The data indicate that H19 modulates Wnt signaling pathway through miR-22; miR-22 could partially reverse the effect of H19 on Wnt signaling pathway." Human L cellular senescence 29520849 Sen_G_1154 MIR107 406901 ncRNA HUVEC -- Aging Accelerate Cell cycle analysis "Cell cycle analysis showed a significant increase in the percentage of cell population arrested at G0/G1 phase in the mimic miR-107 transfected cells compared to the mimic negative control cells.Moreover, the percentage of cell population in S-phase was also significantly lower in the mimic miR-107 transfected cells compared to the mimic negative control cells." p16NK4A//PTEN Upregulation//Downregulation Western blot//RNA Pull-down assay//qRT-PCR "Over-expression of miR-107 also resulted in significant augmentation of the expression of P16INK4A .For further verification, miRNA-mRNA complex pull-down assay was performed.Accordingly, qRT-PCR analysis demonstrated a significant enrichment of PTEN mRNA in the pull-down miRNA-mRNA complex . In addition, PTEN protein expression was down-regulated following the over-expression of miR-107 similar to that observed in the 1 nM rapamycin-treated pre-senescent HUVECs." -- -- -- -- Human HL cellular senescence 29857052 Sen_G_1155 RP11-670E13.6 -- ncRNA HDF -- Aging Prevent SA-β-gal activity assay//Knockdown//Flow cytometry//Cell cycle analysis "RP11-670E13.6 expression was knocked down using an siRNA, which significantly decreased the proliferation of HDFs.Our analysis revealed that RP11-670E13.6-depleted HDFs showed elevated SAβ-gal activity, a distinguishing marker of senescent cells, but no significant effects on apoptosis were observed.RP11-670E13.6 knockdown significantly inhibited cell cycle progression, as evidenced by a significant increase in the G0/G1 fraction and a significant decrease in G2/M cells." -- -- -- -- p16-pRb -- Western blot//RT-PCR "RP11-670E13.6 knockdown increased expression of p16，RP11-670E13.6 knockdown promoted cell cycle arrest and senescence, perhaps through the p16-pRB pathway, independently of the p53-p21 pathway。Knockdown of RP11-670E13.6 had a significant effect on cell senescence-associated mRNA expression. Knockdown of RP11-670E13.6 had a significant effect on cell senescence associated protein expression." Human HL cellular senescence 29143326 Sen_G_1156 MIR138-1 406929 ncRNA SN-12 -- Renal carcinoma Accelerate SA-β-gal activity assay "Ninety-six hours after introducing the miR-138 mimic, the results revealed that overexpression of miR-138 significantly increased the number of SA-β-gal-positive cells compared with those of the scrambled miRNA mimic." E2H2 Downregulation Luciferase reporter assay//SA-β-gal activity assay//Knockdown The transfection of this reporter construct in the presence of miR-138 mimic induced nearly a 50% downregulation of relative luciferase activity; this effect was abolished with the mutation of miR-138 at the putative target site.Knockdown of EZH2 induced senescence in SN-12 cells. -- -- -- -- Human HL cellular senescence 24406044 Sen_G_1157 MIR22 407004 ncRNA EPC -- Aging Accelerate SA-β-gal activity assay//MTT assay "Using β-galactosidase expression as an indicator of cell senescence, we found that overexpression of miR-22 in Y-EPCs increased senescence.Overexpression of miR-22 in Y-EPCs inhibited proliferation." Akt3 Downregulation Luciferase reporter assay//Western blot//RT-PCR "miR-22 overexpression decreased the relative luciferase activity of the 3′UTR reporter, whereas it had no effect on the mutant 3′UTR reporter, indicating that the intact seed sequence is required for miR-22 functionality.miR-22 overexpression in Y-EPCs significantly downregulated AKT3 mRNA and protein expression." -- -- -- -- Human L cellular senescence 25323119 Sen_G_1158 MIR181A1 406995 ncRNA CD4+T -- HCV infection Accelerate SA-β-gal activity assay//EdU assay "MiR-181a reconstitution significantly reduced the telomere length, increased SA-β-gal+ T cell frequency, and decreased EdU incorporation, in CD4+ T cells from patients with HCV." SIRT1 Downregulation Flow cytometry "Transfection of miR-181a precursor, but not the negative control, led to a significant down-regulation of Sirt1 expression in CD4+ T cells." -- -- -- -- Human L cellular senescence 27354409 Sen_G_1159 MIR503 574506 ncRNA HPNE -- Pancreatic cancer Prevent SA-β-gal activity assay//Flow cytometry//Colony formation assay "miR-503 mimic significantly promoted anchoragedependent colony formation, accelerated cell cycle progression, and decreased KRASG12D-induced senescence in HPNE/KRASG12D/shNC cells.miR-503 inhibitor significantly inhibited anchorage-dependent colony formation, retarded cell cycle progression, and increased cell senescence in HPNE/KRASG12D/shARID1A." p16 Downregulation Western blot "The expression of p16 was obviously decreased in HPNE/KRASG12D/ shNC cells upon the transfection of miR-503 mimic, whereas miR503 inhibitor led to increased p16 expression in HPNE/KRASG12D/ shARID1A cells, as shown by western blot assay." -- -- -- -- Human L cellular senescence 28942143 Sen_G_1160 MIR146A 406938 ncRNA HUVEC -- Aging Prevent SA-β-gal activity assay "In P5 HUVEC, we found that miR-146a overexpression decreased the percentage of SA-β-gal-positive cells compared with control at 3 days after transfection." -- -- -- -- -- -- -- -- Human L cellular senescence 21511256 Sen_G_1161 MIR10A 406902 ncRNA MSC -- Aging Prevent Knockdown//SA-β-gal activity assay "Down regulation of miR-10a expression increased the percentage of SA-β-gal positive cells among the LV-anti-10a-infected groups compared to the LV-controlinfected groups, indicating that repression of miR-10a increased hMSC senescence in both young and old hMSCs." KLF4 Downregulation Luciferase reporter assay//Western blot//qRT-PCR "Luciferase activity was significantly repressed by the miR-10a mimic, proving the direct binding of the miR-10a to the 30 -UTR of KLF4. A similar effect was also observed in the hMSCs.To determine if miR-10a would affect endogenous KLF4 expression, we compared KLF4 expression after infecting the hMSCs with LV-miR-10a and LVanti-10a. Up regulation of miR-10a repressed both the endogenous mRNA and protein expression of KLF4." -- -- -- -- Human HL cellular senescence 23696417 Sen_G_1162 MIR34A 407040 ncRNA HASMC -- Cardiovascular disease Accelerate SA-β-gal activity assay//Western blot//Flow cytometry "In comparison with the control (miRNA mimic control [SCR]), miR-34a mimic reduced cell number already by 48 hours after transfection;the main and statistically significant differences were observed at 72- hours post-transfection. Further, we evaluated cell cycle distribution by fluorescence-activated cell sorting analysis after propidium iodide (PI) staining. miR-34a ectopic expression significantly increased the percentage of HASMCs in G0–G1 phase at 24-hours post-transfection when compared with negative control.Accordingly, p21 protein levels were higher in miR-34a-overexpressing cells.We found that miR-34a enhanced the percentage of SA-β-gal-positive cells at 72 hours after transfection, while HASMCs transfected with the negative control did not show any difference when compared with untransfected cells ." SIRT1 Downregulation Western blot "We then performed a rescue experiment by transfecting replicative cells with miR-34a mimic or mimic negative control along with either a 3?-untraslated region-deleted-SIRT1-expression vector (devoid of the sequences matching miR-34a seed sequence) or the corresponding empty vector. The WB analysis confirmed that endogenous SIRT1 protein levels were severely lowered upon miR-34a overexpression, while exogenous SIRT1 protein levels were unaffected." -- -- -- -- Human L cellular senescence 25352462 Sen_G_1163 MIR152 406943 ncRNA HDPSC -- Aging Accelerate SA-β-gal activity assay//MTT assay "The MTT assay showed that miR152 upregulation significantly decreased the viability of HDPSCs.Furthermore, we found that miR-152 overexpression significantly increased the number of SA-β-gal-positive cells concomitant with a significant increase in p21 and p16 expression levels compared with vector control cells." SIRT7 Downregulation Knockdown//Western blot//qRT-PCR//Luciferase reporter assay "Transduction of late-passaged HDPSCs with a lentiviral vector encoding anti-miR-152 effectively downregulated miR-152 and upregulated SIRT7.Luciferase activity assays showed that miR-152 significantly decreased luciferase activity in cells transfected with the wild-type but not the mutant SIRT7 3'-UTR-binding site, confirming that SIRT7 is a direct target of miR-152. Real time RT-PCR and western blot analysis showed that SIRT7 was significantly downregulated in HDPSCs at PD54 compared with its expression at PD16, and significantly downregulated by ectopic expression of miR-152, further supporting SIRT7 as a direct target of miR-152." -- -- -- -- Human L cellular senescence 26991832 Sen_G_1164 MIR21 406991 ncRNA U87 -- Glioma Prevent SA-β-gal activity assay//Knockdown//MTT assay "Compared with the blank and NC groups, the miR-21 mimics and siRNA-SPRY1 groups revealed a decreased density of U87 cells and enlarged cell size, in addition to reduced senescence indexes. In the miR-21 inhibitors group, the senescence index of U87 cells was increased; and no significant difference in senescence index was identified in the miR-21 inhibitors + siRNA-SPRY1 group.MTT results indicated that, compared with the blank and NC groups, cell proliferation reduced in the miR-21 inhibitors group and increased in the miR-21 mimics and siRNA-SPRY1 group; there were no significant differences in cell proliferation in the miR-21 inhibitors + siRNA-SPRY1 group." SPRY1 Downregulation Dual-Luciferase reporter assay//qRT-PCR "The results of the dual-luciferase reporter assay system indicated that there was a significant reduction in the luciferase signal in SPRY1-wt cotransfection group, compared with other miR-21 transfection groups. And, there was no difference in the luciferase signal of SPRY1-mut among miR-21 transfection groups.The results of RT-qPCR showed that, compared with the control group, mRNA levels of miR-21, PI3K, and AKT increased in the glioma group, but mRNA levels of SPRY1 and PTEN decreased." PTEN-PI3K-AKT -- Western blot//RT-PCR "In the miR-21 mimics and siRNA-SPRY1 groups, protein levels of PI3K, AKT, p-AKT, P53, and p-GSK3 increased, and those of SPRYI1, PTEN, Caspase-3, and Caspase-9 decreased;and the miR-21 inhibitors group exhibited opposite trends; in the miR-21 mimics group, miR-21 expression and mRNA expressions of PI3K and AKT increased while mRNA levels of SPRY1 and PTEN decreased ." Human L cellular senescence 29316313 Sen_G_1165 MIR449A 554213 ncRNA "95D,A549" -- Lung cancer Accelerate EdU assay/SA-β-gal activity assay//Flow cytometry "MiR-449a introduction caused a remarkable inhibition of cell growth in both A549 and 95D cells relative to NC. The EdU incorporation assay also revealed that the growth of A549 and 95D cells was significantly inhibited 231 by miR-449a relative to NC . The EdU cell proliferation as- 232 say determined that miR-449a suppressed the entry of A549 and 233 95D cells into the S phase.The miR-449a mimics caused significant G0/G1 arrest in A549 and 95D cells. We also observed that miR-449a in A549 and 95D cells caused senescent phenotypes with positive staining for senescence-associated β-galactosidase (SA-β-gal) and led to drastic changes in cell morphology, including increased size and a broad, flattened shape 72 h following transfection." E2F3 Downregulation Dual-Luciferase reporter assay//qRT-PCR//Western blot "The luciferase reporter assay indicated that the luciferase activity of the re porter containing the E2F3 gene’s wide-type 3' -UTR decreased (52%) following treatment with miR-449a mimics. By contrast, the inhibitory effect of the miR-449a mimics was abolished in the mutated construct. In addition, qRT-PCR and western blot analysis revealed that the expression of E2F3 mRNA and protein was inhibited by treatment with miR-449a mimics in A549 and 95D cells." -- -- -- -- Human L cellular senescence 24211326 Sen_G_1166 MIR340 442908 ncRNA GIC -- Glioma Accelerate BrdU assay/Western blot//SA-β-gal activity assay//Flow cytometry "We observed that miR-340-overexpressing cells became flatter and larger than control cells.Immunohistochemical analysis and Western blot assays revealed that miR-340 overexpressing hGICs partially decreased Nestin expression and lost the expression of Sox2, but remained positive for GFAP, an astrocyte marker. miR-340-overexpressing hGICs ceased proliferating during the first 3 days of culture. The BrdU-incorporation and cell cycle analyses revealed that miR-340 overexpression significantly arrested the cell cycle at the G1/S transition, as indicated by a marked accumulation of cells in the G1 peak and by a reduction of cells in the S phase. We further demonstrated that miR-340 overexpression activated the expression of SA-β-gal, a marker of cellular senescence, in hGICs." PLAT Downregulation Luciferase reporter assay//Western blot//Immunohistology "Immunohistochemical analysis and Western blot assays confirmed that miR-340 overexpression decreased PLAT expression in hGICs. Using a reporter vector encoding the firefly luciferase gene with the wild-type plat 3’UTR, we demonstrated that miR-340 overexpression inhibited luciferase activity in hGICs, whereas a deletion in the predicted binding site of miR-340 in the 3’UTR of the plat gene abrogated the aforementioned inhibitory effect of miR-340. Taken together, these data strongly indicate that PLAT is a novel direct target of miR-340 in hGICs." -- -- -- -- Human L cellular senescence 25627976 Sen_G_1167 MIR34A 407040 ncRNA "UM-SCC-23,Fadu" -- Squamous cell carcinoma Accelerate SA-β-gal activity assay//Flow cytometry "Compared with the wide type and control cells, the miR-34a over-expression cells exhibited an about 1.6 to 2.0 times increase in the percentage of cell at G0/G1 phase, along with a?decreased cell percentage at the S?phase.The result showed that the mean percentage of SA-β-gal-positive cells increased by 4.41 times (UM-SCC-23-miR-34a) and 8.18 times (Fadu-miR-34a) in miR-34a over-expression cell lines than that in control cell lines." MAP2K1//AXL//FUT1//AREG --//--//--//-- Luciferase reporter assay//qRT-PCR "The result indicated that the mRNA levels of 8 genes were reduced about 1.4-fold to 4.7-fold in cells with ectopic miR-34a expression, compared with control.The luciferase reporter activity of pSiCheck?-2-AXL-3’UTR, pSiCheck?-2-FUT1-3’UTR and pSiCheck?-2-MAP2K1-3’UTR was markedly reduced by forced expression of miR-34a compared with the control(2.43- fold decrease, 2.08-fold decrease and 4.78-fold decrease,respectively), whereas no reporter activity were found when their target sites were mutated." -- -- -- -- Human HL cellular senescence 28485160 Sen_G_1168 MIR125B1 406911 ncRNA "KYSE150,KYSE510" -- Esophageal cancer Accelerate CCK-8 assay//SA-β-gal activity assay//Transwell assay "Overexpression of miR-125b-5p significantly inhibited cell proliferation of KYSE150 and KYSE510 cell lines.Using Senescence β-Galactosidase Staining Assay, we found that overexpression of miR-125b-5p significantly enhanced the senescence of ESCC cells. We also found that miR-125b5p overerxpression significantly inhibited the migration and invasion of ESCC cells." HMGA2 Downregulation Western blot//qRT-PCR//Knockdown//Dual-Luciferase reporter assay "HMGA2 was one of the candidate targets, and down-regulated by miR-125b-5p mimics both in mRNA and protein levels. Dual-luciferase reporter assays found that luciferase activity was significantly down-regulated in the miR-125b-5p mimics and pGL3-HMGA2-3’UTR-Wt group compared with NC group." -- -- -- -- Human HL cellular senescence 28968424 Sen_G_1169 MIR34A 407040 ncRNA PPC-1 -- Prostate cancer Accelerate SA-β-gal activity assay//Flow cytometry MiR-34a overexpression induced significantly increased cell senescence assessed by staining of prostate cancer cells for senescence-associated β-gal (SA-βgal) activity. It is well documented that G1 cell-cycle arrest generally precedes cell senescence. -- -- -- -- -- -- -- -- Human L cellular senescence 22719071 Sen_G_1170 MIR212 406994 ncRNA HCT116 -- Prostate cancer Accelerate SA-β-gal activity assay "In both SF and CM conditions, we show that transfection with miR-212 resulted in increased cellular senescence as shown by senescence associated β-galactosidase staining as compared to negative control mimic." SIRT1 Downregulation SA-β-gal activity assay "We asked, whether miR-212 could modulate cellular senescence. Using HCT116 cells, we demonstrate that miR-212 inhibit endogenous SIRT1 levels in both serum free (SF) and complete media (CM). Co-transfection with SIRT1 reduced the number of senescent cells in miR-212 transfected cells partially abrogating its senescence inducing affect." -- -- -- -- Human L cellular senescence 26439987 Sen_G_1171 MIR23A 407010 ncRNA HDF -- Aging Accelerate SA-β-gal activity assay//qRT–PCR//Histochemical staining//FACS analysis "Non-senescent fibroblasts were transfected with the miR-23a-3p mimic and oligonucleotide control. Seven days after the transfection, a threefold increase in SA-β-gal was detected.Inhibition of mir-23a-3p in senescent fibroblasts reduced the senescent phenotype of the cells and rescued the loss of the HA matrix." HAS2 Downregulation qRT-PCR//Luciferase reporter assay "Quantitative real-time reverse-transcriptase–PCR (qRT-PCR) analysis revealed that miR-23a-3p expression was increased and HAS2 mRNA expression decreased in senescent compared with non-senescent fibroblasts.In skin biopsies from mice aged 18 and 48 weeks, the miR-23a-3p copy number was increased and the HAS2 mRNA expression decreased, as seen in aged and senescent human dermal fibroblasts previously. Transfection of senescent cells, which display high levels of endogenous miR-23a-3p, with the miR-23a-3p inhibitor increased the HAS2 mRNA copy number twofold. Luciferase activity was not decreased when cells were transfected with luciferase constructs containing a mutated version of the HAS2 3′UTR binding site and the miR-23a-3p mimic ." -- -- -- -- Human HL cellular senescence 25264594 Sen_G_1172 MIR141 406933 ncRNA MSC Umbilical cord blood Aging Accelerate SA-β-gal activity assay//MTT assay "In the miR-141-3p-transfected cells, MTT assays and cell cycle analyses revealed that the growth rate was reduced 48?hours post-transfection. In contrast, the transfection of hMSCs with anti-miR-141-3p yielded an increase in proliferation. The correlation between the expression of ZMPSTE24 and miR-141-3p was confirmed in five other hMSC cell lines .The overexpression of miR-141-3p also increased SA-β-gal activity and γH2Ax expression in these cells." ZMPSTE24 Downregulation Western blot//Knockdown "We further investigated whether miR-141-3p regulates ZMPSTE24 expression, prelamin A accumulation and nuclear abnormalities. The transfection of hMSCs with miR-141-3p decreased ZMPSTE24 expression, resulting in increased expression of prelamin A, p16INK4A?and γH2AX. Conversely, these effects were reversed by miR-141-3p knockdown." -- -- -- -- Human L cellular senescence 24101728 Sen_G_1173 MIR17 406952 ncRNA MCF Heart Aging Prevent SA-β-gal activity assay//Cell apoptosis assay//qRT-PCR "Transfection of the miR-17-3p inhibitor enhanced MCF senescence and apoptosis in cells treated with H2O2, whereas expression of miR-17-3p repressed senescence and apoptosis.Consistent with these results, we found that the aging hearts expressed significantly lower levels of miR-17-3p compared with the levels in hearts from young animals." Par4 Downregulation Luciferase reporter assay//Western blot//SA-β-gal activity assay "Luciferase activity was repressed when the construct (Luc-Par4) was co-transfected with miR-17-3p mimic, and the repression was abolished when the target site was mutated, thus confirming direct targeting of Par4 by miR-17-3p. Protein lysates prepared from miR-17- and vector-transfected cells were subjected to western blotting, which showed that ectopic transfection of miR-17 repressed Par4 expression significantly. Ectopic expression of Par4 promoted senescence when the cells were cultured in serum-free medium or treated with H2O2." CEBPB-FAK -- Western blot//IP//RT-PCR//PCR//ChIP "The ChIP assay indicated that Par4 might bind to the CEBPB promoter directly and repress CEBPB transcription, whereas CEBPB could bind to the FAK promoter and enhance FAK transcription. By using western blotting, we confirmed that silencing Par4 promoted the expression of CEBPB and FAK.The Par4-expressing cells showed high levels of Par4 and low levels of CEBPB and FAK, and the regulation appeared to occur at the transcriptional level. In addition, ectopic transfection of CEBPB enhanced FAK expression at the mRNA and protein levels. By ChIP, immunoprecipitated exogenous Par4 was shown to pull down more CEBPB promoter DNA, whereas immunoprecipitated exogenous CEBPB could pull down more FAK promoter DNA." Human L cellular senescence 25472717 Sen_G_1174 MIR143 406935 ncRNA VSMC -- Aging Accelerate SA-β-gal activity assay "Our results showed that overexpression of miR-143 promoted senescence,and miR-143 inhibitor suppressed senescence induced by H2O2." -- -- -- -- -- -- -- -- Human L cellular senescence 25655189 Sen_G_1175 MIR449A 554213 ncRNA "B5,PC-3,DU 145,DU-1.1" -- Prostate cancer Accelerate SA-β-gal activity assay//Flow cytometry "We transfected DU-145 sublines with miR-449a and stained for SA-β-gal activity. At 10 and 25 nM concentrations, DU-1.1 and B5 cells stained positive for SA-β-gal, while staining in mock and miR-Con treatments were nearly undetectable . In PC-3 cells, miR-449a caused G0/G1 arrest as indicated by the increase in G0/G1 cell number and corresponding reductions in S and G2/M populations." Cyclin D1//HDAC1 Downregulation//Downregulation Western blot//Knockdown miR-449a significantly reduced Cyclin D1 protein levels in PC-3 cells.Knockdown of HDAC1 by miR-449a or siHDAC1 elevated p27 protein and reduced P-Rb levels. Rb Downregulation Western blot//Knockdown Knockdown of Cyclin D1 by miR-449a or siCCND1 drastically reduced phophorylated Rb (P-Rb) levels. Knockdown of HDAC1 by miR-449a or siHDAC1 elevated p27 protein and reduced P-Rb levels. Human L cellular senescence 20948989 Sen_G_1176 MIR21 406991 ncRNA HUVEC -- Aging Accelerate SA-β-gal activity assay//MTT assay//qPCR "Stable miR21 over-expression indeed significantly reduced cell proliferation consistent with the transient over-expression results above.Cell growth was further monitored during the entire replicative lifespan, and indeed, miR-21 over-expressing cells underwent replicative senescence earlier than control cells.Furthermore, miR-21 over-expression led to an increase in senescent cells, as the majority of cells stain positive for senescenceassociated ?-galactosidase (SA ?-gal) at PD1pT after selection of stable cells." CDC25A//NF1B --//-- Western blot "CDC25A and NF1B are direct targets of miR‐21. The expression of a luciferase reporter gene containing the CDC25A 3′‐UTR (A) or NF1B 3′‐UTR (B), respectively, was suppressed by miR‐21 in HUVEC. The suppression is specific to the miR‐21 seed region within the CDC25A 3′‐UTR or NF1B 3′‐UTR, as mutation of the miR‐21 target sites alleviated repression by miR‐21. Ratios of CDC25A‐ or NF1B‐dependent renilla luciferase signal (rLuc) to a firefly luciferase signal (fLuc) encoded by the same vector are given." -- -- -- -- Human HL cellular senescence 23496142 Sen_G_1177 MIAT 440823 ncRNA MCF-7 -- Breast cancer Prevent SA-β-gal activity assay//Knockdown//Flow cytometry//Western blot "The results showed an increase in the proportion of cells in G1 phase and a decreased in the proportion of cells in G2/M phase in Miat knock down cells.The result revealed that Cyclin D1 expression level was downregulated in cells treated with Miat siRNA in comparison to cells treated with scramble siRNA, which indicated Miat inhibition caused cell cycle arrest. The result revealed that the proportion of senescent cells was dramatically elevated in Miat knockdown cells in comparison to control cells. Consistent with above stain positive for senescence-associated β-galactosidase activity, the expression of cellular senescent biomarkers, p16Ink4A and Cox2, markedly increased following Miat suppression." -- -- -- -- -- -- -- -- Human L cellular senescence 29345338 Sen_G_1178 OVAAL 148756 ncRNA "ME4405,HCT116" -- Colorectal cancer Prevent Knockdown//PI staining//Flow cytometry//SA-β-gal activity assay "OVAAL shRNA inhibited cancer cell proliferation, which was substantiated through G0/G1 cell cycle arrest and decreased phosphorylated Rb (p-Rb); depletion of OVAAL also triggered cellular senescence as shown by induction of senescence-associated (SA)–?-gal staining ." STK3//p27 Binding//-- Western blot//qPCR//RIP "Further examination of the relationship between OVAAL and p27 expression showed that the increased p27 protein levels observed after silencing of OVAAL did not increase p27 mRNA levels.Consistently, OVAAL was coimmunoprecipitated by STK3 antibodies by RNA immunoprecipitation (RIP) assays in ME4405 and HCT116 cells ." Raf-MEK-ERK Activation Western blot "Silencing of OVAAL prominently reduced the activation of RAF/MEK/ERK cascade as shown by decreases in p-MEK, p-ERK, p-MSK1, and p-RSK1, recapitulating the effects observed after knockdown of STK3 in ME4405 and HCT116 cells." Human L cellular senescence 30478051 Sen_G_1179 MIR34A 407040 ncRNA TIG3 TERT/B-RAF:ER -- Aging Accelerate Cell cycle analysis//Cell morphological analysis Cells overexpressing miR-34a exhibited a senescence-like morphology.Overexpression of miR-34a reduced cellular proliferation. B-RAFoncogene//ELK1//Myc --//Binding//Downregulation qPCR//Knockdown//Western blot "miR-34a was approximately eightfold upregulated after 3 days of B-RAF activation. ER cells using siRNA and measured miR-34a regulation after B-RAF activation，depletion of ELK1 significantly impaired B-RAF-mediated induction of miR-34a(P<0.04, Student's t-test). Strikingly, miR-34a overexpression resulted in marked downregulation of endogenous MYC protein." -- -- -- -- Human HL cellular senescence 19696787 Sen_G_1180 MIR137 406928 ncRNA IMR-90 -- Pancreatic cancer Accelerate SA-β-gal activity assay//Immunostaining "A robust senescence response was observed upon transfection of miR-137 compared to miR-67-transfected cells, as assessed using senescence-associated β-galactosidase activity .an increase in the number of PML nuclear bodies and γH2AX foci, two known markers of cells undergoing cellular senescence, also were observed in miR-137-expressing cells." KDM4A Downregulation qRT-PCR//Western blot "The mRNA level of CHD5, a transcriptional target of KDM4A (Mallette and Richard, 2012), was increased following transfection with miR-137, consistent with decreased KDM4A mRNA expression and protein levels." ARF-p53//p16-pRb Activation//Activation SA-β-gal activity assay//qRT-PCR "Expression of human papilloma viral E6 and E7 in miR-137-transfected fibroblasts fully rescued the senescence phenotype, suggesting that miR-137 indeed relies on either the p53 or p16INK4A?pathway to establish senescence." Human L cellular senescence 26904954 Sen_G_1181 MIR494 574452 ncRNA HUVEC -- Aging Accelerate SA-β-gal activity assay//Cell cycle analysis//Western blot//Cell morphological analysis "Gain of miR-494 in early-passage HUVECs increased senescence-associated β-galactosidase levels (SA-β-gal).Ectopic expression of miR-494 increased G1 arrest, whereas the inhibition of the miR decreased the G2 arrest in response to a 10?Gy dose of radiation .miR-494 impacted other hallmarks of senescence—a decrease in telomerase activity , an increase in the cell cycle regulator p21 as well as a decrease in Rb hyperphosphorylation .phalloidin staining in HUVECs after 48?h of miR-494 treatment also revealed flattened and multinucleated cells." MRN Downregulation qRT-PCR//Western blot//Immunostaining//Luciferase reporter assay "Transfection of miR-494 decreased both MRN RNA and protein levels,miR-494 decreased activity of a luciferase reporter cloned upstream of each 3’-UTR of the MRN complex genes . miR-494-transfected HUVECs also showed a decrease in MRE11a nuclear foci." -- -- -- -- Human L cellular senescence 29795397 Sen_G_1182 MIR221 407006 ncRNA A549 -- Lung cancer Accelerate SA-β-gal activity assay "A549-miR-221 cells induced cellular senescence following Cisplatin treatment as compared to A549-Cont cells, detected by SA-β-gal activity assay." -- -- -- -- PTEN-Akt -- Western blot "When miR-221 was ectopically expressed, PTEN was decreased and Akt activity was increased in H1299 cells. On the other hand, miR-221 antisense oligonucleotides (ASO) were used to suppress miR-221 activity, and upregulation of PTEN and downregulation of Akt activity in A549 cells were accompanied." Human L cellular senescence 29876362 Sen_G_1183 TERC 7012 ncRNA -- Lung Inflammation Prevent SA-β-gal activity assay//Western blot "β-Gal staining for cellular senescence was markedly increased in G3 TERC-null lung sections compared with control;We analyzed the levels of heterochromatin protein 1γ(HP1γ), a marker for senescence-associated heterochromatin foci, and found that immunoreActivate HP1γ was also markedly increased, with the level of HP1γ in TERC-/- AECII being more than 4 times that in WT control." -- -- -- -- -- -- -- -- Human L cellular senescence 26518879 Sen_G_1184 MIR29C 407026 ncRNA MSC -- Aging Accelerate SA-β-gal activity assay//EdU assay//CCK-8 assay//qRT-PCR "The results demonstrated that the over-expression of miR-29c-3p significantly enhanced the SA-β-gal staining compared with that of the negative control (NC) group. EdU was not incorporated into the SA-β-gal positive hMSCs, indicating the senescent hMSCs were indeed not proliferating.the CCK-8 assay was performed ,The results showed that the proliferative capacity of hMSCs was inhibited after the Agomir-29c-3p transfection.The qPCR results indicated that p16, p21, PTGS2, AKAP9, CCND1 and EDN1(senescence markers ) were significantly increased after miR-29c-3p transfection." CNOT6 -- Western blot//qRT-PCR "We transfected hMSCs with Agomir-29c-3p or mirVana-29c-3p and examined CNOT6 expression using qPCR and WB assays. The results showed that the CNOT6 mRNA level was decreased in the Agomir-29c-3p group, but increased in the mirVana-29c-3p group." p53-p21//p16-pRb Upregulation//Upregulation Western blot//qRT-PCR "Western blot (WB) results also suggested that the expression of p53, p21, p16, PTGS2 and pRB were all increased gradually." Human L cellular senescence 26792405 Sen_G_1185 MIR1236 100302242 ncRNA A549 -- Lung cancer Accelerate SA-β-gal activity assay//Colony formation assay Positive β-galactosidase cells were observed to increase prominently following transfection of the miR-1236.miR-1236 was obviously suppressed the number of colonies. p21 Upregulation Flow cytometry//SA-β-gal activity assay//Colony formation assay Flow cytometry analysis suggested that cotransfection of miR-1236 with siP21 remarkably attenuated Go/G1 cycle arrest.Silencing p21 dramatically inhibited miR-1236 induced cell senescence.The ability of proliferation and colony formation were restored by p21 silencing. CCND1-CDK4//CCND1-CDK6 Downregulation qRT-PCR "As is shown, compared to dsControl treatment, positive control (dsP21-322), miR-1236-3p significantly downregulated expression of Cyclin D1 and CDK4/CDK6 mRNA in A549 ." Human L cellular senescence 28631573 Sen_G_1186 MIR370 442915 ncRNA A549 -- Lung cancer Accelerate SA-β-gal activity assay//Colony formation assay Positive β-galactosidase cells were observed to increase prominently following transfection of the miR-370.miR-370 was obviously suppressed the number of colonies. p21 Upregulation Flow cytometry//SA-β-gal activity assay//Colony formation assay Flow cytometry analysis suggested that cotransfection of miR-370 with siP21 remarkably attenuated Go/G1 cycle arrest.Silencing p21 dramatically inhibited miR-370 induced cell senescence.The ability of proliferation and colony formation were restored by p21 silencing. CCND1-CDK4//CCND1-CDK6 Downregulation qRT-PCR "As is shown, compared to dsControl treatment, positive control (dsP21-322), miR-370 significantly downregulated expression of Cyclin D1 and CDK4/CDK6 mRNA in A549 ." Human L cellular senescence 28631573 Sen_G_1187 MIR21 406991 ncRNA HUVEC -- Aging Accelerate SA-β-gal activity assay The percentage of β-galactosidase staining-positive cells in the ox-LDL group was significantly increased compared to that in the control group. The ox-LDL-induced endothelial cell senescence was markedly reduced in the presence of miR-21-5p inhibitor. The negative control of inhibitor did not exert effect on ox-LDL-induced endothelial cell senescence. Drp1 Downregulation Western blot//qRT-PCR "Both mRNA and protein expressions of Drp1 were significantly downregulated in the ox-LDL-treated HUVECs, which were reversed in the presence of miR-21-5p inhibitor." AMPK-p53//AMPK-p16 Activation Western blot "The phosphorylation level of AMPK(p-AMPK) was obviously elevated in the ox-LDL-treated HUVECs compared to that in the control cells, concomitant with an up-regulation of p53 and p16, indicating an activation of AMPK-p53/p16 pathway.These phenomena were blocked by miR-21-5p inhibitor." Human L cellular senescence 28347692 Sen_G_1188 MIR203A 406986 ncRNA HUVEC -- Aging Accelerate SA-β-gal activity assay The percentage of β-galactosidase staining-positive cells in the ox-LDL group was significantly increased compared to that in the control group. The ox-LDL-induced endothelial cell senescence was markedly reduced in the presence of miR-203a-3p inhibitor. The negative control of inhibitor did not exert effect on ox-LDL-induced endothelial cell senescence. Drp1 Downregulation Western blot//qRT-PCR "Both mRNA and protein expressions of Drp1 were significantly downregulated in the ox-LDL-treated HUVECs, which were reversed in the presence of miR-203a-3p inhibitor." AMPK-p53//AMPK-p16 Activation Western blot "The phosphorylation level of AMPK(p-AMPK) was obviously elevated in the ox-LDL-treated HUVECs compared to that in the control cells, concomitant with an up-regulation of p53 and p16, indicating an activation of AMPK-p53/p16 pathway.These phenomena were blocked by miR-203a-3p inhibitor." Human L cellular senescence 28347692 Sen_G_1189 H19 283120 ncRNA HUVEC -- Cardiovascular disease Prevent SA-β-gal activity assay//Western blot//Flow cytometry//Knockdown "We first analyzed the role of H19 in proliferation. siRNA-mediated depletion of H19 in HUVECs led to a significant reduction of cells in S- and G2/M-phase, while cells accumulated in G0/G1 phase;silencing of H19 increased the number of acidic β-galactosidase positive HUVECs and hCoAECs, which is a marker of cellular senescence;Lentivirus-mediated overexpression of H19 tended to reduce p16 and p21 expression;These findings demonstrate that H19 negatively regulates the well-described increase in inflammatory activation in aging." -- -- -- -- STAT3 Downregulation qRT-PCR "STAT3 is known to regulate p21 and ICAM-1,the inhibition of STAT3 with Cryptotanshinone (CPT) abolished the H19 depletion-mediated induction of p21. Furthermore, the induction of ICAM-1 and VCAM-1 after H19 depletion was attenuated by STAT3 inhibition as well ." Human HL cellular senescence 30107531 Sen_G_1190 MALAT1 378938 ncRNA Fibroblast Foreskin Aging Accelerate SA-β-gal activity assay "The ratio of senescent cells was markedly greater following UVB irradiation (74.4%) compared with cells that had not been exposed to irradiation (15.7%).In addition, the ratio of senescent cells was significantly lower following intervention with MALAT1 siRNA (52.1%) compared with the UVB irradiation group." -- -- -- -- ERK-MAPK -- Western blot MALAT1 siRNA inhibited UVB-induced ERK phosphorylation. Human L cellular senescence 28487970 Sen_G_1191 MIR30C1 407031 ncRNA BJ Pancreas Aging Prevent Cell proliferation assay//SA-β-gal activity assay//qRT-PCR//Growth curve assay Increased expression of each of the 5 miR-30 family members abrogated induction of proliferative arrest and SA-β-gal by HarasV12 in BJ cells .We detected less SA-β-gal positive epithelial cells in PanIN lesions from the miR-30c-1 transgenic than the control pancreas. CHD7//TNRC6A//p16//p53 Downregulation//Downregulation//Downregulation//Downregulation qRT-PCR "All 5 miR-30 members inhibited the expression of endogenous CHD7 and TNRC6A mRNA in BJ cells;Corresponding to reduced senescence in the skin tumors from miR-30c transgenic mice, these tumors contained less cells positive for γH2AX or activated p53 (p53-pS15), indicating abrogation of DDR, and less p16INK4A-positive cells, as compared to those from wild-type littermates ." -- -- -- -- Human L cellular senescence 29907771 Sen_G_1192 MIR34A 407040 ncRNA "U87,MSC" -- Aging Accelerate qRT-PCR//CCK-8 assay//SA-β-gal activity assay//ELISA "Co-culturing with MSCs overexpressing miR-34a for 24 h impaired U87 glioma cell proliferation. Additionally, it induced expression of senescence-associated genes p53, Cdkn1a and Cdkn2c. As expected,this result was corroborated by an increase in the percentage of cells positively marked with the senescence-associated β-galactosidase. It was identified that co?culture with hMSCs overexpressing miR-34a decreased the telomere length and impaired the telomerase activity." SIRT1 Downregulation Western blot "The results confirmed that the level of SIRT1 decreased in U87 cells co-cultured with hMSCs overexpressing miR-34a, while NC mimic transfection had no observable effect." -- -- -- -- Human L cellular senescence 30592284 Sen_G_1193 MIR34A 407040 ncRNA ADSC -- Aging Accelerate CCK-8 assay//Western blot//SA-β-gal activity assay "The miR-34a treated cells exhibited significant reduction in cell proliferation in comparison with the scrambled miR control group.miR-34a overexpression in ADSCs induced a significant decrease in expression of cyclins and CDKs.In contrast, miR-34a treatment induced the increased expression of the CDK inhibitors(P53,P21) in ADSCs.The number of SA-β-gal-stained cells was markedly increased by miR-34a overexpression and recovered byco-treatment with anti-miR-34a, indicating the strong induction of cellular senescence by miR-34a." -- -- -- -- -- -- -- -- Human HL cellular senescence 26677981 Sen_G_1194 MIR377 494326 ncRNA HSF -- Aging Accelerate SA-β-gal activity assay//MTS assay//Western blot "Moreover, overexpression of miR-377 increased the SA-β-gal-positive ratio and p16 expression while decreased HSF proliferation in young HSFs." DNMT1//p53 Downregulation//Upregulation qRT-PCR//Western blot "Computational miRNA target analysis from a miRNA database demonstrated that miR-377 had high homology with a sequence in the 3′-UTR of human DNMT1 mRNA.DNMT1 mRNA and protein expression levels were significantly decreased by the miR-377 mimics and elevated by the miR-377 inhibitors.Of these three genes, p53 was further confirmed to be regulated by miR-377 using RT-qPCR and western blotting. Increased p53 mRNA,p53 and Rb expressions together with diminished phosphoryla- tion of Rb were induced by the miR-377 mimics and reverse changes were induced by the miR-377 inhibitors compared with that of the respective controls." -- -- -- -- Human HL delay aging 28277545 Sen_G_1195 MIR15B 406949 ncRNA HDF -- Aging Prevent qRT-PCR "We observed an increased expression at day 2 upon transfection of miR-15b inhibitors followed by a clear decrease of transcript levels (31-80%) at day 3 (IFNγ) and day 4 (IL-1α, IL-1β, IL-6, IL-8, and VEGF)." SIRT4 Downregulation qRT‐PCR//Luciferase reporter assay "Similarly, and again in accordance with the SIRT4 expression status,miR-15b levels were also decreased in UV- irradiated or gamma-irradiated senescent primary human dermal fibroblasts.In these experiments, miR-15b mimics dampened basal SIRT4 mRNA levels and inhibited senescence-associated upregulation of SIRT4 transcripts.Moreover, by employing luciferase assays we show that the 3’UTR of human SIRT4 is a direct target of miR-15b, as miR-15b mimics significantly inhibited the relative luciferase activity of a transfected SIRT4 - 3’-UTR construct, but not, when the SIRT4 – 3’-UTR was mutated within the seed sequence for miR-15b." -- -- -- -- Human HL delay aging 26959556 Sen_G_1196 MIR1292 100302138 ncRNA Adipose stromal cell -- Age-related osteoporosis Accelerate qRT-PCR//Western blot//SA-β-gal activity assay "qRT-PCR and western blot analyses also showed that lenti-1292-infected hADSCs expressed higher levels of the senescence markers P16 and P21 and lower expression of LMNB1 than control cells.Moreover, the number of SA-β-gal-stained cells was increased after miR-1292 overexpression." FZD4 Downregulation Luciferase reporter assay//qRT-PCR//Western blot "Results showed that miR-1292 overexpression remarkably inhibited luciferase reporter activity from the vector containing the WT FZD4 3′ UTR, but not from the mutated FZD4 or those of other genes. Additionally, after miR-1292 overexpression, FZD4 was markedly down -regulated at the protein level, but not at mRNA level, Also, qRT-PCR results showed that the expression of miR-1292 was negatively correlated with FZD4 in 70 clinical bone samples." Wnt-β-catenin Downregulation Western blot "Western blotting studies revealed that miR-1292 overexpression decreased both FZD4 and β-catenin expression, indicating that Wnt/β-catenin signaling was impaired." Human L delay aging 30574422 Sen_G_1197 CircPVT1 -- ncRNA WI-38 -- Aging Prevent SA-β-gal activity assay//Cell morphological analysis//Western blot Silencing CircPVT1 led to increased levels of senescence marker TP53(p53) and triggered a flattened and enlarged cell morphology accompanied by increased SA-β gal activity. let-7 Downregulation qRT-PCR//SA-β-gal activity assay//Cell morphological analysis//Western blot//Dual-Luciferase reporter assay "One of the microRNAs,let-7, was found to be the most highly enriched microRNA in the CircPVT1 pulldown.Importantly, silencing CircPVT1, which increases let-7 availability, significantly reversed this loss of P21 and TP53 expression observed with anti-let-7 alone.SA-βgal activity was elevated in CircPVT1-silenced cells, while anti-let-7 had the opposite effect, reducing SA-βgal activity and increasing proliferation; importantly, the proliferative phenotype seen after antagonizing let-7 was rescued when CircPVT1 was silenced. Although pre-let-7-transfected cells showed greater reduction in psiCHECK2-let-7 activity(RL/FL) (45% of control), silencing CircPVT1 also significantly lowered RL/FL activity (70% of control),indicating that CircPVT1 reduced the availability of functional let-7 to transcripts bearing a let-7 site." -- -- -- -- Human HL delay aging 27928058 Sen_G_1198 MEG3 55384 ncRNA HUVEC Blood Aging Prevent Flow cytometry//FISH//Knockdown "The si-MEG3 + platelet group exhibited further elevated ROS production compared with the si-MEG3 group. Results of telomere length determination showed that telomere length was reduced by MEG3 silencing, and this reduction was enhanced by MEG3 silencing and platelets." -- -- -- -- -- -- -- -- Human L delay aging 30576236 Sen_G_1199 MIR15B 406949 ncRNA "ES‐2,Caov‐4,SKOV3,OVCAR-3,OV‐1063" -- Ovarian cancer Accelerate MTT assay//Flow cytometry//SA-β-gal activity assay "Cell proliferation, migration and invasion abilities in addition to cell proportion in the S phase in response to miR-15b mimic and siRNA LPAR3 significantly decreased while apoptosis and senescence, as well as cell proportion in G0/G1 phase significantly elevated (p < 0.05)." LPAR3 Downregulation Dual-Luciferase reporter assay "The luciferase reporter exhibited that in comparison with the NC group, the luciferase activity of LPAR3 WT was profoundly reduced by miR 15b (p < 0.05), whereas that of the LPAR3 MUT was not changed. " PI3K-Akt Downregulation Western blot//qRT-PCR "In relative to the blank and NC groups, the miR-15b and Bax expression in ovarian cancer cells treated with miR 15b mimic dramatically enhanced (p < 0.05), but that of LPAR3 and Bcl 2, as well as the extent of PI3K and Akt phosphorylation, remarkably diminished (p < 0.05)." Human HL delay aging 31140597 Sen_G_1200 MIR183 406959 ncRNA "HTM,HDF,HeLa" -- Aging Accelerate BrdU assay Forced expression of miR-183 resulted in significant inhibition of cell proliferation in both primary HTM cells and HDFs but had no effect on the proliferation of HeLa cells. -- -- -- -- -- -- -- -- Human HL delay aging 27116545 Sen_G_1201 MIR675 100033819 ncRNA H9C2 -- Vascular disease Prevent SA-β-gal activity assay//EdU assay "The data showed that miR-675 mimic increased the expression of miR-675 and miR-675 inhibitor decreased the expression of miR-675.Importantly, the miR-675 mimic decreased β-gal staining and increased the numbers of proliferating cells, while the miR-675 inhibitor showed an opposite effect on β-gal staining and cell proliferation." -- -- -- -- TGFβ1-p21 Downregulation Luciferase reporter assay//qRT-PCR//Western blot "Dual-luciferase reporter assays showed that the miR-675 mimic decreased the luciferase activity compared to the control. To confirm that TGF-β1 is a target of miR675 in H9C2 cells, we transfected miR-675 mimic or inhibitor into these cells. We found that miR-675 mimic and inhibitor did not alter the expression of TGF-β1 mRNA, but miR-675 mimic decreased the expression level of TGF-β1 protein and miR-675 inhibitor has the opposite effect. These findings verified TGF-β1 as the target gene of miR-675. Moreover, p21 protein level was reduced by miR-675 mimic, but increased by miR-675 inhibitor." Human L delay aging 30889706 Sen_G_1202 LNCTAM34A 102724571 ncRNA HCT116 Colon Aging Prevent SA-β-gal activity assay//Knockdown//Cell viability assay "In addition, depletion of GUARDIN triggered cellular senescence.The effects of GUARDIN on cell viability were mirrored in the long-term survival of HCT116 cells in clonogenic assays and in their growth in nu/nu mice." -- -- -- -- p53 -- Western blot//Cell viability assay//Knockdown "Although GUARDIN knockdown in the absence of p53 did not significantly affect cell viability, cell death was enhanced following induction of p53 . Conversely, overexpression of GUARDIN diminished the reduction in cell viability associated with p53 induction. Thus, although p53 controls the function of GUARDIN through regulating its expression, GUARDIN modulates the cytotoxic effect of p53.We extended our analysis to multiple lines, confirming that overexpression of wild-type p53 in U2OS osteosarcoma, A549 lung adenocarcinoma and HCT116 cells increased GUARDIN levels, whereas knockdown of p53 decreased GUARDIN expression." Human HL delay aging 29593331 Sen_G_1203 MIR21 406991 ncRNA MCF-7 -- Breast cancer Prevent SA-β-gal activity assay//Knockdown//MTT assay//ELISA "Pre-miR-21 stimulated MCF-7 cell proliferation at the time points considered. In contrast, significant growth inhibition by ATRA was observed upon miR-21 silencing, indicating sensitization of MCF-7 cells. The effect of miR-21 silencing on ATRA-induced senescence was evaluated, using two molecular markers: -galactosidase and trimethyl K9 histone H3." IL1B//ICAM-1//PLAT Downregulation//--//Downregulation Luciferase reporter assay//qRT-PCR//Western blot "We cloned the 3-UTR of the selected transcripts downstream of a luciferase reporter and evaluated the effect of miR-21 in 293T cells, which contain low levels of the miRNA (54) .MiR-21 inhibited the expression of the ICAM-1, PLAT, and IL1B constructs, indicating that they are direct targets.Forced expression of pre-miR-21 reduced ATRA-dependent induction of PLAT and IL1B mRNAs and proteins. By converse, the mRNA or protein expression pattern of ICAM-1 observed in ATRA-treated MDA-MB-231 cells was not affected by pre-miR-21 transfection. In contrast, pre-miR-21 enhanced the down-regulation of maspin mRNA and protein afforded by ATRA." -- -- -- -- Human HL delay aging 21131358 Sen_G_1204 ACSL5 51703 ncRNA "RT4,RT-112,Urotsa" Tumor tissue Bladder cancer Accelerate SA-β-gal activity assay "Senescence-associated-β-galactosidase activity was significantly associated with ACSL5 upregulation in tumours (p=0.03) and highest activity was also found in RT4 cells. RT112 and Urotsa showed no β-galactosidase activity, and in J82 enzyme activity was minimal." -- -- -- -- -- -- -- -- Human L delay aging 23348389 Sen_G_1205 MIR146A 406938 ncRNA "HTM636,HTM1073" -- Aging Prevent SA-β-gal activity assay//BrdU assay//Flow cytometry "Production of SA-β-gal and iROS was significantly decreased, and BrdU incorporation was significantly increased by miR-146a in both HTM cell lines." PAI-1//IRAK1 --//-- BrdU assay "We investigated whether the observed effects of miR-146a on SA-β-gal activity, iROS, and cell proliferation in senescent HTM cells were mediated by the downregulation of either the direct target IRAK1 or the secondary target PAI-1.Overexpression of both PAI-1 and IRAK1, lacking the miR-146a target site, inhibited the effects of miR-146a on cell proliferation." -- -- -- -- Human HL delay aging 20053980 Sen_G_1206 MIR138-1 406929 ncRNA "HuH-7,HepG2,Hepatocyte" -- Hepatocellular carcinoma Accelerate SA-β-gal activity assay//BrdU assay "Telomeric repeat amplification protocol (TRAP) assay, senescence-associated β-galactosidase assay and BrdU incorporation assay were performed 24 and 48 h after transfection. The results showed that miR-138 functioned to decrease telomerase activity, which in turn induced cell senescence and suppressed cell proliferation." TERT Downregulation Western blot//qRT-PCR//Luciferase reporter assay "The binding site of miR-138 in the 3’UTR of TERT gene was predicted by miRNA target prediction tools. The results showed that wild-type miR-138, but not mutant miR-138 (mutated in the seed-complementary sequence),can significantly suppress the luciferase activities of Huh7 cells transfected with wild-type TERT 3’UTR construct,Quantitative real-time PCR and Western blotting analysis showed that wild-type miR-138 mimic can suppress endogenous TERT mRNA and protein expression in Huh7 and HepG2 cells. These results indicated that TERT can be a direct target of miR-138 in HCC cells." -- -- -- -- Human L delay aging 28258280 Sen_G_1207 MIR101-1 406893 ncRNA MCF-7 -- Aging Accelerate SA-β-gal activity assay "Further, continuous (upto 72 hrs) over-expression of miR-101 depicted an enhanced induction of senescence(2.3-Fold±0.25). miR-101 inhibition with anti-miR-101 oligonu-cleotides depicted a profile (0.9-Fold 60.25) which matched with the control cells." UBE2N//SMARCA4 Downregulation//Downregulation qRT-PCR "The validation of the two targets, UBE2N and SMARCA4, was established by measuring their endogenous cellular expression as well, which decreased significantly in over-expressing miR-101 cells." -- -- -- -- Human HL delay aging 25353636 Sen_G_1208 HOTAIR 100124700 ncRNA "WI-38,IDH4" -- Aging Accelerate qRT-PCR//SA-β-gal activity assay//Flow cytometry "The levels of senescent markers p53, p21 and p16 were elevated, and cells displayed the characteristic senescence- associated (SA) increase in G1 cell cycle compartment.That the increased HOTAIR was important for the implementation of senescence was evidenced by experiments in which silencing HOTAIR in IDH4 cells reversed the senescent phenotype, with a lower proportion of cells displaying the hallmark SA-β-galactosidase activity along with reduced levels of senescence markers." Ataxin-1//Snurportin-1 Downregulation//Downregulation Western blot//Co-IP "Interestingly,the interaction of Dzip3 and Ataxin-1, as well as the interaction of Mex3b and Snurportin-1, as assessed by co-IP followed by western blot (WB) analysis, was facilitated by HOTAIR overexpression; furthermore, the levels of Ataxin-1 and Snurportin-1 decreased after HOTAIR overexpression." -- -- -- -- Human L delay aging 24326307 Sen_G_1209 MALAT1 378938 ncRNA "GBC-SD,SGC-996" -- Gallbladder cancer Prevent SA-β-gal activity assay//EdU assay//Transwell assay//Western blot "Our results indicated that when MALAT1 was silenced, the viability of the GBC-SD and SGC-996 cells was suppressed, and the expression of Ki67 and PCNA was reduced. Meanwhile, the capabilities of migration and invasion were inhibited, while the SA-β-gal activity was promoted (p? 4-fold and > 5-fold, respectively, in mutant compared to control or SMAD4-WT cells. Consistent with this, expression of the proliferative marker Ki67 was substantially reduced in SMAD4-R496C mutant compared to control cells. Quantitative analysis of the cell cycle regulators, CDK2 and CDC25A, indicated reduced expression in SMAD4-R496C compared to controls, providing further evidence for repressed or halted cell cycle progression and enforcement of cellular senescence." -- -- -- -- TGF-β/SMAD-IFNγ Activation qRT-PCR//SA-β-gal activity assay "Consistent with this, the growth curve of Werner cells displayed an increased doubling time with passage compared to control normal fibroblasts. At passage 14, the population doubling time of Werner cells was markedly increased compared to corresponding controls, suggesting proliferative arrest and accelerated senescence of Werner cells, as described previously. Increased senescence of Werner cells was further supported by increased numbers of SA-beta positive cells and expression of IL-6, STAT1, and IFNγ." Human L cellular senescence 33428109 Sen_G_1306 MT-CO2 4513 protein coding HMSCs -- Aging Prevent WST-1 assay//SA-β-gal activity assay The results of WST-1 assay revealed that the COX2 transfectants grew at a faster rate than the normal HMSCs. SA-β-Gal staining revealed that the overexpression of COX2 significantly delayed the aging process of HMSCs. -- -- -- -- -- -- -- -- Human L cellular senescence 33416107 Sen_G_1307 MIR217 406999 ncRNA HUVECs -- Aging Accelerate SA-β-gal activity assay "The data showed that overexpression of miR-217 significantly increased the proportion of SA-β-gal positive cells in PDL12, PDL16 and PDL20 cells" SIRT1 Downregulation qRT-PCR//Western blot "Western blot and RT-qPCR results showed that up-regulation of miR-217 in PDL8 cells significantly inhibited SIRT1 expression, whereas miR-217 inhibitor significantly inhibited SIRT1 expression in PDL44 cells." SIRT1-p53 Activation qRT-PCR//Western blot "SIRT1 protein levels and mRNA levels were significantly lower in senescent PDL44 HUVECs than in young PDL8 HUVECs, while p53 protein levels and mRNA levels were significantly higher in senescent PDL44 HUVECs than in young PDL8 HUVECs" Human L cellular senescence 33392891 Sen_G_1308 TWIST1 7291 protein coding NP Cells Nucleus pulposus Disc degeneration Prevent Western blot//SA-β-gal activity assay//qRT-PCR//CCK-8 assay "Of note, in the TWIST overexpression group, the p53 and p21 protein were effectively suppressed compared to the TNF-α group. In addition, the collagen II and β-gal protein expression were determined by IF staining. However, TWIST1/2 overexpressed NP cells were partly efficient in inhibiting the MMP-13 and IL-1β upregulation. The cell proliferation was determined by CCK-8 assay, and the result indicated the TWIST1/2 overexpressed NP presented a higher proliferative ability than the non-transfected cells under the treatment of TNF-α." -- -- -- -- p53-p21 Downregulation Western blot "Of note, in the TWIST overexpression group, the p53 and p21 protein were effectively suppressed compared to the TNF-α group. Besides, the p53 and p21 levels were inhibited due to the supplement of TWIST protein." Human L cellular senescence 33378011 Sen_G_1309 TWIST2 117581 protein coding NP Cells Nucleus pulposus Disc degeneration Prevent Western blot//SA-β-gal activity assay//qRT-PCR//CCK-8 assay "Of note, in the TWIST overexpression group, the p53 and p21 protein were effectively suppressed compared to the TNF-α group. In addition, the collagen II and β-gal protein expression were determined by IF staining. However, TWIST1/2 overexpressed NP cells were partly efficient in inhibiting the MMP-13 and IL-1β upregulation. The cell proliferation was determined by CCK-8 assay, and the result indicated the TWIST1/2 overexpressed NP presented a higher proliferative ability than the non-transfected cells under the treatment of TNF-α." -- -- -- -- p53-p21 Downregulation Western blot "Of note, in the TWIST overexpression group, the p53 and p21 protein were effectively suppressed compared to the TNF-α group. Besides, the p53 and p21 levels were inhibited due to the supplement of TWIST protein." Human L cellular senescence 33378011 Sen_G_1310 CDK2 1017 protein coding Phoenix-Eco -- Leukemia Prevent SA-β-gal activity assay//Western blot//Immunohistochemistry//Immunofluorescence "Although samples from both CVT2584- and vehicle-treated mice showed a certain degree of positivity, the SA-β-gal staining was significantly increased in spleens from CVT2584-treated mice, as quantitated in Figure 5. We also examined the presence of senescence-associated heterochromatin foci (SAHF), using an antibody directed against H3K9me3. Immunofluorescence staining of spleen tissue from CVT2584-treated mice showed a strong increase in SAHF formation compared to the vehicle-treated mice. Nuclear p19ARF-positive cells were readily detected in sections of CVT2584-treated animals but found virtually absent in the analyzed specimens from vehicle-treated animals. Nuclear p19ARF-positive cells were readily detected in sections of CVT2584-treated animals but found virtually absent in the analyzed specimens from vehicle-treated animals." MYC Upregulation Western blot "In agreement with our previous report, reduced MYC Ser-62 phosphorylation was observed in CVT2584-treated compared with vehicle-treated animals, both at lower and higher doses of CVT2584, supporting the notion that CDK2 regulates MYC phosphorylation in vivo." -- -- -- -- Human L cellular senescence 33356836 Sen_G_1311 CFL1 1072 protein coding WI-38 -- Aging Accelerate SA-β-gal activity assay//Western blot//Immunofluorescence "Subsequently, we assessed the expression of the cell cycle inhibitors involved in senescence‐associated growth arrest. We found that the over‐expression of cofilin‐1 in young cells could induce p53, p21Cip1, p27Kip1, p16INK4, and p‐cofilin‐1, but the silencing of cofilin‐1 in senescent cells suppressed these molecules. Subsequently, we assessed the expression of the cell cycle inhibitors involved in senescence‐associated growth arrest. We found that the over‐expression of cofilin‐1 in young cells could induce p53, p21Cip1, p27Kip1, p16INK4, and p‐cofilin‐1, but the silencing of cofilin‐1 in senescent cells suppressed these molecules. We then found that the levels of SA‐β‐gal stained cells were increased by the over‐expression of cofilin‐1 in young cells and were decreased by the knockdown of cofilin‐1 in senescent cells. " TEAD1 Downregulation qRT-PCR "To investigate whether the down‐regulation of TEAD1 mRNA is associated with up‐regulated cofilin‐1 in senescent cells, we silenced cofilin‐1 and found that the TEAD1 mRNA levels were restored in senescent cells. Additionally, the over‐expression of cofilin‐1 could suppress the expression of TEAD1 transcripts and protein in H1299/tet‐on‐cofilin‐1 cells. " -- -- -- -- Human L cellular senescence 33336885 Sen_G_1312 GATA6 2627 protein coding iPSC-MSC -- Aging Accelerate SA-β-gal activity assay//qRT-PCR "GATA6 knockdown in cells at the late passage significantly reduced the ratio of senescent to total cells whereas that in cells at the early passage did not. Similarly, GATA6 knockdown markedly decreased the expression of p53 and p21CIP1 and significantly increased the expression of CDK1 in late-passage cells but did not affect early-passage cells. Other than the effect on cell cycle regulators, GATA6 knockdown significantly downregulated mRNA levels of senescence-associated secretory phenotype (SASP) marker IL6 and IL1B in late-passage as well as early-passage iPSC-MSCs." -- -- -- -- SHH-FOXP1 Downregulation SA-β-gal activity assay//Western blot "Knockdown of GATA6 resulted in an increase in the expression of the SHH signaling molecule SHH, SMO, and GLI1 as well as FOXP1, suggesting that GATA6 is an upstream inhibitory regulator of both SHH signaling and FOXP1. When the expression of SMO, a key molecule in the SHH pathway, was repressed, the expression of its downstream molecule GLI1 was decreased as well as that of FOXP1. There was a significantly lower ratio of cells stained positive for SA-β-gal in the culture of GATA6-knockdown cells compared to that in the culture of control cells whereas the opposite trend was shown between the culture of FOXP1-knockdown and control cells, suggesting that repressing the expression of GATA6 or FOXP1 leads to attenuation or increase of cellular senescence, respectively. Results of DNA content analysis showed that repressing the expression of GATA6 in iPSC-MSCs promoted cell proliferation whereas that of FOXP1 significantly inhibited cell proliferation. In terms of the activity of senescence- and cell cycle-associated markers, GATA6 knockdown resulted in a significant decrease in the expression of p53 and p21CIP1 and a significant increase in the expression of CDK1. Conversely, FOXP1 knockdown significantly increased the p53 and p21CIP1 expression and significantly decreased the CDK1 expression." Human HL cellular senescence 33252174 Sen_G_1313 PDK1 5163 protein coding NHDF -- Aging Accelerate SA-β-gal activity assay//qRT-PCR//EdU assay "Either PDK1 inhibition or dual inhibition of mTOR and NF-κB reduced the SA-β-gal–positive cell population from >80% to <40%, whereas mTOR inhibition alone did not significantly alter the percent of SA-β-gal–positive cells. We found that the percent of proliferating cells increased after either PDK1 inhibition or dual inhibition of mTOR and NF-κB, but not after only mTOR inhibition. Senescent cells showed a significantly increased expression of these SASP-associated genes. Either exposure to the NF-κB inhibitor JSH23 or the PDK1 inhibitor BX795 reduced the expression of these genes. " mTOR//NF-κB Upregulation qRT-PCR//EdU assay " We found that the percent of proliferating cells increased after either PDK1 inhibition or dual inhibition of mTOR and NF-κB, but not after only mTOR inhibition. Senescent cells showed a significantly increased expression of these SASP-associated genes. Either exposure to the NF-κB inhibitor JSH23 or the PDK1 inhibitor BX795 reduced the expression of these genes. " -- -- -- -- Human HL cellular senescence 33229519 Sen_G_1314 SLX4IP 128710 protein coding "DU145,PC-3" -- CRPC Prevent SA-β-gal activity assay//Western blot "As expected, a considerable increase in the proportion of senescence-associated β-galactosidase (SA β-gal) staining was observed in late-passage cells with SLX4IP knockdown compared with late-passage controls. Elevations in relative p21 protein expression, a senescence-associated marker, further corroborated the senescent phenotype. Moreover, SLX4IP knockdown led to a prominent increase in both β-gal positively stained cells and relative p21 protein expression in late-passage DU145-derived cell lines that were not present in early-passage cells." -- -- -- -- -- -- -- -- Human L telomere attrition 33188147 Sen_G_1315 MEG3 55384 ncRNA "A549,MCF-7" -- Aging Accelerate SA-β-gal activity assay//Western blot//qRT-PCR "The degree of senescence of A549 and MCF-7 cells was detected by SA-β-gal staining after depletion of lncRNA MEG3, showing a significant decrease compared with that of the control group. Next, we induced senescence by etoposide after overexpression of lncRNA MEG3 in A549 and MCF-7 cells, and increased expression of p21 and p16 was observed in senescent models.The mRNA and protein expression levels of senescence-related markers were attenuated to some extent after depletion of lncRNA MEG3 in senescent models of A549 and MCF-7 cells, indicating that lncRNA MEG3 might play an important role in the regulation of senescence induced by etoposide." miR-16-5p Binding Dual-Luciferase reporter assay "The results showed that the luciferase activity decreased significantly in Hek293T cells co-transfected with wild type lncRNA MEG3 and miR-16-5p mimics, whereas luciferase activity was restored in cells co-transfected with mutant lncRNA MEG3 and miR-16-5p mimics, indicating that miR-16-5p is indeed a direct target gene of lncRNA MEG3 and lncRNA MEG3 plays a vital role in the senescent process by acting as a ceRNA." miR-16-5p-VGLL4 Activation qRT-PCR//SA-β-gal activity assay "Moreover, silencing lncRNA MEG3 or VGLL4 significantly alleviated low dose etoposide induced senescence, as demonstrated by decreased p21 and p16 expression and SA-β-gal staining positive cells. Interestingly, the effect of the inhibition of lncRNA MEG3 on the senescence of tumor cells was prominently abolished by the miR-16-5p inhibitor, as suggested by increased p21 and p16 expression and SA-β-gal staining positive cells." Human HL cellular senescence 33141998 Sen_G_1316 NEAT1 283131 ncRNA BM-MSC -- Aging Prevent SA-β-gal activity assay//qRT-PCR "QRT-PCR results suggested that exosomeMIF transferred LncRNA–NEAT1 to cardiomyocytes, while silencing LncRNA–NEAT1 in MSCs blocked the transfer process. In the subsequent experiments, exosomeMIF significantly reduced the percentage of cells in the G0/G1 phase, expression of p27 and p16, and number of SA-β-gal-positive cells. However, it elevated the telomere length and activity." miR-221-3p Binding Dual-Luciferase reporter assay//qRT-PCR//SA-β-gal activity assay "LncRNA–NEAT1 contains the binding site for miR-221-3p, suggesting that lncRNA as a ceRNA sponged miR-221-3p to limit its function, which was confirmed by dual-luciferase gene reporter assay. As illustrated in Fig. 6b, the co-transfection of miR-221-3p significantly inhibited the luciferase activities elicited by LncRNA–NEAT1. Further, a biotin–avidin pulldown system was employed to test whether miR-221-3p could pull down NEAT1. Cardiomyocytes were transfected with biotinylated miR-221-3p, then collected for biotin based pulldown assay. NEAT1 was pulled down as analyzed by qRT-PCR. Further, a biotin–avidin pulldown system was employed to test whether miR-221-3p could pull down NEAT1. Cardiomyocytes were transfected with biotinylated miR-221-3p, then collected for biotin based pulldown assay. NEAT1 was pulled down as analyzed by qRT-PCR. Importantly, miR-221-3p overexpression markedly increased the number of cells in the G0/G1 phase, expression of p27 and p16, and number of SA-β-gal-positive cells, but inhibited the telomere and telomerase activity, even when exosomeMIF were added to Dox-treated cardiomyocytes." -- -- -- -- Human HL cellular senescence 33129330 Sen_G_1317 HEIH 100859930 ncRNA "OVCA429,OVCA433,OVCAR3" -- Ovarian cancer Prevent SA-β-gal activity assay "As shown in Fig. 2H, the percentage of SA-b-Gal-positive cells was increased by HEIH knockdown in OVCA429 (sh-HEIH#1: 5.2-fold higher than sh-NC group; sh-HEIH#2: 4.6-fold higher than sh-NC group) and OVCA433 cells (sh-HEIH#1: 4.8-fold higher than sh-NC group; shHEIH#2: 4.7-fold higher than sh-NC group)." miR-3619-5p Binding Dual-Luciferase reporter assay//qRT-PCR//RIP "From Figure 3D, the transfection of miR-3619-5p mimics showed no difference on the luciferase activity of pmirGLO-HEIH-Mut vectors, but significantly decreased the luciferase reporter activity of pmirGLO-HEIHWt vectors in OVCA429 (0.33-fold decreased) and OVCA433 cells (0.41-fold decreased) (n ? 3, P < 0.05). In addition, RIP assay indicated that HEIH-Wt and miR-3619-5p were apparently enriched in the Ago2 pellet but not the IgG control pellet in both OVCA429 (HEIH: 4.93-fold increased; miR-3619-5p: 3.21-fold increased) and OVCA433 cells (HEIH: 5.24-fold increased; miR-3619-5p: 3.07-fold increased). RT-qPCR analysis was carried out and the results indicated that HEIH expression was significantly decreased by miR-3619-5p mimics (OVCA429: 0.14-fold decreased; OVCA433: 0.26fold decreased), whereas HEIH knockdown markedly increased miR-3619-5p expression (OVCA429: 3.24-fold increased; OVCA433: 2.87-fold increased)." miR-3619-5p-CTTNBP2 Downregulation Dual-Luciferase reporter assay//qRT-PCR//RIP "As presented in Figure 5C, the luciferase reporter activity of pmirGLO-CTTNBP2-Wt vectors was significantly reduced by miR-3619-5p mimics transfection (OVCA433: 0.38-fold decreased; OVCA429: 0.44-fold decreased). RIP assay indicated that miR-3619-5p (OVCA429: 785-fold increased; OVCA433: 833-fold increased) and CTTNBP2 expression (OVCA429: 1370-fold increased; OVCA433: 1260-fold increased) was significantly enriched in the Ago2 pellet rather than in the IgG control pellet. RT-qPCR and western blot analyses displayed that the mRNA and protein levels of CTTNBP2 were markedly decreased by miR-3619-5p overexpression (OVCA429: 0.15-fold decreased; OVCA433: 0.34-fold decreased) or HEIH knockdown (OVCA429: 0.22-fold decreased; OVCA433: 0.37-fold decreased). Additionally, the percentage of SA-b-Gal-positive cells was increased by HEIH knockdown, whereas these effects were abrogated by CTTNBP2 overexpression in OVCA429 (5.2-fold higher than sh-NC group; 0.2-fold lower than sh-HEIH#1 group) and OVCA433 cells (4.8-fold higher than sh-NC group; 0.2-fold lower than sh-HEIH#1 group)." Human L cellular senescence 33110047 Sen_G_1318 SNHG29 125144 ncRNA HTR8/SVneo -- Preterm birth Accelerate SA-β-gal activity assay//qRT-PCR "SA-β-Gal increased in the SNHG29 + H2O2 cells as compared to the NC + H2O2 cells (7.8%, 15.9% and 23% respectively). We measured the levels of pro-inflammatory cytokines, such as IL-8 and TNF- α. The mRNA levels of IL-8 and TNF-α decreased in cells depleted of SNHG29 compared with NC-Si and NC-Si + H2O2 cells. However, there was an increase in the expression of IL-8 and TNF-α in cells transfected with AAV-SNHG29 compared to NC and NC + H2O2 cells." -- -- -- -- p53-p21 Activation Western blot//SA-β-gal activity assay//qRT-PCR "Treated cells depleted of SNHG29 (si-SNHG29 + H2O2) showed a reduction in the levels of mRNA of p53 and p21, and the protein levels of p53, phospho-p53,p21and phospho-p21, as compared to treated cells with wild-type SNHG29. Overexpression of SNHG29 in H 2O2-treated cells (SNHG29 + H2O2) showed increases in the level of mRNA of p53 and p21, and protein levels of p53, phospho-p53, p21and phospho-p21 as compared to treated cells with wild-type SNHG29. " Human L cellular senescence 33080448 Sen_G_1319 METTL3 56339 protein coding hMSCs -- Progeroid syndromes Prevent SA-β-gal activity assay//Clonal expansion assay "Furthermore, METTL3-deficient hMSCs acquired premature aging phenotypes, as evidenced by decreased proliferative capacity and increased percentage of SA-β-Gal-positive cells. Moreover, enhanced cell proliferative capacity and reduced SA-β-Gal-positive cells were observed in HGPS and WS hMSCs upon METTL3 overexpression." MIS12 Downregulation qRT-PCR//Western blot "Among these transcripts, we noticed that m6A modifications in MIS12, a key regulator of cell proliferation, were markedly reduced in both prematurely senescent and METTL3-deficient hMSCs, as validated by MeRIP-qPCR analysis. We also detected a significant decrease in MIS12 expression at both the mRNA and protein levels in HGPS and WS hMSCs as well as in METTL3-deficient hMSCs. Among these transcripts, we noticed that m6A modifications in MIS12, a key regulator of cell proliferation, were markedly reduced in both prematurely senescent and METTL3-deficient hMSCs, as validated by MeRIP-qPCR analysis. We also detected a significant decrease in MIS12 expression at both the mRNA and protein levels in HGPS and WS hMSCs as well as in METTL3-deficient hMSCs." -- -- -- -- Human HL cellular senescence 33035345 Sen_G_1320 HES1 3280 protein coding Dermal fibroblasts Skin Aging Prevent SA-β-gal activity assay//Clonal expansion assay "Ectopic overexpression or CRISPR-dCas9-mediated endogenous activation of HES1 protected human fibroblasts against aging phenotypes, evidenced by increased cell proliferation and attenuated cell senescence" -- -- -- -- -- -- -- -- Human HL cellular senescence 33238152 Sen_G_1321 KLF6 1316 protein coding Dermal fibroblasts Skin Aging Prevent SA-β-gal activity assay//Clonal expansion assay//qRT-PCR//ELISA "Senescence-associated β-galactosidase (SA-β-gal) staining showed that knockdown of KLF6 promoted senescence of human primary epidermal keratinocytes In addition, the downregulation of KLF6 in human primary keratinocytes led to decreased proliferation ability and increased expression and secretion of pro-inflammatory cytokine IL6, in line with the decreased self-renewal and upregulated cytokine signaling in aged epithelial cells." -- -- -- -- -- -- -- -- Human HL cellular senescence 33238152 Sen_G_1322 PPARA 5465 protein coding "EA.hy926,HUVEC" -- Atherosclerosis Prevent SA-β-gal activity assay//Immunostainings "β-gal staining showed that the number of senescent cells decreased after administration of the PPARα agonist.Immunofluorescence double labeling staining showed that there were a large number of aging endothelial cells in the atherosclerotic plaque of Apoe-/- mice, and the PPARα agonist inhibited the denudation and maintained the integrity of aging endothelial cells in Apoe-/- model mice." GDF11 Binding EMSA//Dual-Luciferase reporter assay "The EMSA results showed that PPARα bound to the labeled target probe, and partially to the mutant probe, indicating that PPARα could bind to the GDF11 target sequence and participate in the regulation of downstream gene transcription. Double-luciferase reporter gene experiment results indicated that PPARα could bind to the GDF11-specific sequence." -- -- -- -- Human L cellular senescence 33728018 Sen_G_1323 SGK1 6446 protein coding Glial cell -- Parkinson disease Accelerate RNA-seq//SA-β-gal activity assay//ELISA//CCK-8 assay "Second, our RNA‐seq data show that treating glia with an SGK1 inhibitor downregulated the pro‐oxidant Cyba, Ncf2, Recql4, Bax, and Fdxr and senescence‐associated secretory phenotype (SASP) genes. We further observed that senescence‐associated β‐galactosidase (SA‐β‐gal) activity, the hallmark senescence biomarker, was promoted in glia exposed to H2O2 and significantly rescued in the presence of the SGK1 inhibitor.In addition, in a paraquat‐induced glial senescence model, a WB analysis showed that the herbicide‐induced LMNB1 (Lamin B1) loss, a senescence‐associated biomarker, was alleviated in glial cultures treated with the SGK1 inhibitor, and levels of the pro‐senescence proteins IL6, MMP3, and p16 (CDKN2A) also decreased. Reduced secretion of IL6, a cytokine of SASP, from cultured glia treated with the SGK1 inhibitor was further demonstrated using ELISA." -- -- -- -- -- -- -- -- Human HL delay aging 33646633 Sen_G_1324 LINC00858 170425 ncRNA HCT116 Colon cancer tissues Colorectal cancer Prevent SA-β-gal activity assay "The results of β-gal staining, immunofluorescence staining, and Western blot analysis demonstrated that the senescence and autophagy of cells treated with sh-NC + sh-WNK2 were reduced in parallel with diminished expression of Beclin-1 and LC3II/I, in contrast to findings in cells treated with sh-NC + sh-NC (p < 0.05). However, opposite effects were observed in cells treated with sh-LINC00858 + sh-WNK2 when compared with sh-NC + sh-WNK2 (p <0.05)." WNK2 Upregulation Western blot "The results from Western blot analysis showed that, compared with cells treated with sh-NC (of WNK2) + sh-NC (of LINC00858), WNK2 expression was reduced in cells treated with sh-NC + sh-WNK2, while its expression was elevated in cells treated with sh-LINC00858 + sh-WNK2 when compared with cells treated with sh-NC + sh-WNK2 (p < 0.05)." -- -- -- -- Human HL apoptosis 32768499 Sen_G_1325 CD70 970 protein coding T cell -- Aging Accelerate Flow cytometry "To investigate the potential role of CD70 signaling in T-cell aging, we examined the expression of CD70 on T cells from 217 healthy adults using flow cytometry. The results showed that CD70-expressing CD4+ and CD8+T cells accumulated with aging.The TCM, TEM, and TEMRA subsets of both CD4+ and CD8+ T cells, known as antigen-experienced T cells, expressed higher levels of CD70 than TN cells regardless of age. Also, CD70 expression was substantially increased in each T cell subset of CD4+ and CD8+ cells from older subjects as compared to young and middle-aged subjects. Thus, an elevated proportion of CD70+ fractions among CD4+ and CD8+ cells in elderly individuals was not only a result of the higher number of antigen-encountered T cells, but also the age-related increase of CD70 expression." -- -- -- -- -- -- -- -- Human L delay aging 32559178 Sen_G_1326 SETMAR 6419 protein coding "U87MG,SF268" -- Glioblastoma Prevent Cell transfection//SA-β-gal activity assay//Western blot "According to experiments, shRNA (sh1) or siRNA (mixture of 3 siRNAs) mediated knockdown of SETMAR initially induced cell death of RR cells; however, eventually (12 days post induction of shRNA and 10-day post siRNA treatment) the cells ceased to grow and became senescent. Importantly, SETMAR knockdown did not alter the growth pattern of parent cells but specifically halted the proliferation of RR cells, highlighting the dependency of RR cells on SETMAR to escape from senescence." -- -- -- -- -- -- -- -- Human HL delay aging 32458986 Sen_G_1327 CD9 928 protein coding HUVEC -- Aging Accelerate Cell transfection//SA-β-gal activity assay//Flow cytometry//Western blot "To examine the roles of CD9 in endothelial cell senescence, we measured the effects of CD9 knockdown in senescent HUVECs (PD>50). Knockdown of CD9 in senescent cells with siRNA reduced the p53 and p21 levels, representative senescence markers, and the pAKT and pS6K levels, decreased SAβG staining, and caused morphological changes similar to those observed in young cells. CD9 downregulation increased cell proliferation, BrdU incorporation, and Ki67 immunoreactivity, which are all well-known cell proliferation markers. CD9 knockdown decreased the G0/G1 cell population and increased the S and G2/M cell population, suggesting a release of G1 arrest, which is a typical phenotype in cellular senescence Young cells transduced with CD9 adenovirus were enlarged and flattened. Moreover, CD9 upregulation increased SAβG staining, but decreased BrdU incorporation, Ki67 immunoreactivity, the proportion of cells in the S phase, and endothelial tube formation." -- -- -- -- PI3K-AKT-mTOR-p53 Activation Cell transfection//SA-β-gal activity assay//Flow cytometry//Western blot "Therefore, we tested whether the PI3K/AKT-mTOR pathway plays a role in CD9-induced senescence. Pretreatment with LY294002, a specific inhibitor of PI3K, or rapamycin, an inhibitor of mTOR, reduced the levels of p53 and p21 and SA-β-gal staining induced by CD9 overexpression. We next tested whether PIK3CA 110β or PIK3CB 110β, two PI3K catalytic subunits, plays a role in CD9-induced senescence. Knockdown of PIK3CA, but not that of PIK3CB, significantly decreased the levels of pAKT, p53, p-p53, pS6K, and p21 proteins, as well as SA-β-gal staining, suggesting that PIK3CA might be involved in CD9-induced cellular senescence. These results indicate that the PI3K-AKT-mTOR-p53 pathway might regulate CD9-mediated cellular senescence." Human L cellular senescence 32346137 Sen_G_1328 FBP1 2203 protein coding HSC -- Aging Accelerate SA-β-gal activity assay//Flow cytometry//γH2AX staining "Importantly, SA-β-Gal+ cells colocalized with those positive for α-SMA (an activated HSC maker), and IL6 (prominent SASP component), and significant percentage of α-SMA+?HSCs also expressed the DNA damage marker γ-H2AX, collectively supporting the presence of senescent HSCs. As DEN is not usually fibrogenic in mice22,24, we surmised that FBP1 deficiency in and of itself promotes HSC activation and senescence. Indeed, liver fibrosis was also detected in?non-DEN treated Cre livers, together with a population of α-SMA+and Ki67+/α-SMA+HSCs." HMGB1 Upregulation IF//Immunoblotting "We therefore treated DEN/GFP or DEN/Cre animals with inflachromene (ICM), a small molecule shown to block HMGB1 release45. ICM treatment greatly reduced not only surface tumours, but also microscopic lesions in DEN/Cre mice. ICM did not affect hepatic steatosis, based on comparable TG levels. As expected, cytosolic HMGB1 levels decreased while nuclear HMGB1 levels increased in ICM-treated livers. ICM also substantially reduced numbers of HSCs expressing SASP components." -- -- -- -- Human HL cellular senescence 32367049 Sen_G_1329 WISP1 8840 protein coding Chondrocyte Cartilage tissues Osteoarthritis Prevent IHC//SA-β-gal staining//FITC-Annexin V/PI "Normal cartilage specimen ?showed very low expression levels of WISP1. The ?protein levels of WISP1 were significantly increased within ?the middle and deep regions of all detected pathological ?cartilage sections.The optical density analyses showed that OA pa?tients appeared to exhibit a higher WISP1 expression in joint ?cartilage than that in control groups ( p < 0.05); the WISP1 ?expression was higher ( p < 0.05) within moderate OA com?pared with that within mild OA; and WISP1 expression in ?severe OA was shown to be remarkably elevated than mod?erate OA ( p < 0.05).The senescence cell percentage and apoptotic cell percentage were ?remarkably downregulated in OA chondrocytes treated with ?rhWISP1 compared with the controls. " -- -- -- -- -- -- -- -- Human L apoptosis 33646053 Sen_G_1330 PPAR 5465 protein coding -- Great saphenous vein wall Chronic venous disorder Prevent RT-qPCR//Immunohistochemical staining "We studied the gene and protein expression of α, β/δ and γ PPAR isoforms in venous wall from CVeD patients and HV controls. We observed a significant decrease in PPAR-α gene expression by RT-qPCR in CVeD patients with respect to HV controls (HV=5.224 [3.659-8.039] RQ vs CVeD=4.632 [2.365-6.387] RQ, ***p=0.0004). We also studied PPAR-α protein expression by immunohistochemistry (IHC). We observed a significant decrease in the score, with a mean of 1.500 [0.500-2.750] in the HV group and 0.000 [0.500-2.750] in the CVeD group, **p=0, 0001. Furthermore, The percentage of positive samples was 100% (n=27) in the HV group and 74.286% (n=26) in the CVeD group." PPAR-α//PPAR-β/δ//TFEB Downregulation//Downregulation//Downregulation Immunohistochemical staining//qPCR We observed a significant ?decrease in PPAR-α gene expression by RT-qPCR in ?CVeD patients with respect to HV controls.//The score for ?PPAR-β/δ was significantly lower for patients with ?CVeD.//Our results showed that the gene expression of ?TFEB was significantly decreased in CVeD patients ?compared with the HV group. -- -- -- -- Human HL cellular senescence 33645625 Sen_G_1331 RECQL4 9401 protein coding "KYSE30,KYSE450,KYSE410,KYSE150,TE-1" ESCC tissues Esophageal cancer Accelerate Western blot//SA-β-gal activity assay "Approximately 45% of KYSE30 RECQL4-depleted cells and 30% of TE-1 RECQL4-depleted cells showed positive SA-β-gal staining. The expression of p21, an effector molecule of G1 arrest and cellular senescence, was also increased significantly, which is consistent with previous studies." c-myc//cyclinD//CDK6//cyclinE Downregulation//Downregulation//Upregulation//Upregulation Western blot "To further investigate how RECQL4 regulates G1/S phase ?transition and cellular senescence, we determined the expres?sion of other related proteins. Depletion of RECQL4 led to decreased expression of c-myc, cyclin D, ?CDK6, and cyclin E. " -- -- -- -- Human HL cellular senescence 33628589 Sen_G_1332 ATM 472 protein coding HFMs Human haired scalp Aging Prevent IHC//Western blot//Cell viability assay "The intensity and the distribution level of the staining of TRP-1 and GP100 was higher in the hair ?follicle melanocytes of fully pigmented follicles, sporadic in melanocytes with reduced pigmentation and lack?ing in weakly pigmented/white hair follicles.Results showed increased total ATM levels in HFMs afer H2O2 treatment, and this increase was blocked by pre?incubation of HFMs with antioxidants VitE/Q for 1h.Both Menadione and H2O2 treatment ?signifcantly reduced cell viability versus vehicle controls.As seen in Fig. 5, results showed that in the absence of ATM kinase activity, sustained H2O2 treatment for 24 h significantly reduced cell viability compared to controls." -- -- -- -- -- -- -- -- Human L apoptosis 33128003 Sen_G_1333 SENEX 93663 protein coding OCI‐LY8 -- r/r DLBCL Accelerate SA-β-gal activity assay "Interestingly, compared with the control group (C group) and scramble negative control (NC group), the senescence rate and proliferation rate were significantly decreased (Figure 5C–G), but the apoptosis rate was significantly increased in SENEX silenced group (SS group)." -- -- -- -- p16-Rb Activation Western blot "We found that p16 ?and pRB expression levels were both significantly increased after the induction of doxorubicin, while RB ?expression level was decreased. " Human L apoptosis 33015970 Sen_G_1334 HBP1 26959 protein coding NP Cells -- Intervertebral disc degeneration Accelerate RT-qPCR "The upregulation of HBP1 also af?fected collagen II expression and contributed to ?the rise of p16 expression.The ?HPB1 also triggered the IL-6, TNF, and MMP-3 ?expressions compared to the control. However, the silencing of HBP1 restrained the p16 expression and protected the collagen II level secreted by NP cells." -- -- -- -- -- -- -- -- Human L apoptosis 32964956 Sen_G_1335 BRD4 23476 protein coding KYSE450 -- Oesophageal cancer Prevent SA-β-gal activity assay//Western blot "Treated with 125nM JQ1 for 6 days, the SA-β-gal?positive cells significantly rose and increased subsequently in a ?dose-dependent manner.Immunoblotting also demonstrated that ?JQ1 increased p21 protein level within 6 days in KYSE450 cells." AURKA//AURKB Upregulation Western blot//ChIP-Qpcr Western blot analysis also showed that AURKA and AURKB protein were time- and ?dose-dependently decreased following JQ1 treatment. Data of ChIP-qPCR ?revealed that JQ1 suppressed BRD4 binding on the promoters of ?AURKA and AURKB genes in KYSE450 cells. -- -- -- -- Human L apoptosis 32954665 Sen_G_1336 MDM2 4193 protein coding MCF-7 -- Breast ?cancer Accelerate SA-β-gal activity assay We observed a significant increase in β- ?galactosidase staining in cells treated with the combination of 1μM NVP-CGM097 and 100 nM fulvestrant for ?72 h compared to vehicle treatment or treatment with ?either single agent alone (p < 0.01 vs NVP-CGM097; p < ?0.001 vs fulvestrant). -- -- -- -- -- -- -- -- Human HL apoptosis 32787886 Sen_G_1337 ASIC1 41 protein coding NP-MSCs -- Aging Accelerate Western blot//CCK-8 assay//Flow cytometry//SA-β-gal activity assay//ELISA "ASIC1 and ASIC3 were progressively upregulated in NP-MSCs derived from mild,moderate through to severely degenerated IVDs. CCK-8 showed that Pfirrmann grade II and grade IV isolates failed to grow at pH 6.6 in contrast to normal culture conditions.proliferative cells (S+G2/M phases) substantially declined from 25.72 to 7.12%.Acidic culture resulted in a time dependent increase in the percentage of senescence cells,reaching a striking ~80% of cells after 5 days.NP-MSC increased levels of secreted IL-6 and IL-8 by NP-MSCs under pH 6.6 culture conditions." p53//p21//p27//Rb1//p16//MMP3//MMP9 Upregulation//Upregulation//Upregulation//Upregulation//Upregulation//Upregulation//Upregulation Western blot "Exposure of isolated NP-MSCs to acidic growth conditions resulted in relatively increased levels of p53,p21,p27,Rb1 and p16 proteins.MMP3 and MMP9 levels also increased whereas aggrecan." -- -- -- -- Human L cellular senescence 33824228 Sen_G_1338 ASIC3 9311 protein coding NP-MSCs -- Aging Accelerate Western blot//CCK-8 assay//Flow cytometry//SA-β-gal activity assay//ELISA "ASIC1 and ASIC3 were progressively upregulated in NP-MSCs derived from mild,moderate through to severely degenerated IVDs. CCK-8 showed that Pfirrmann grade II and grade IV isolates failed to grow at pH 6.6 in contrast to normal culture conditions.proliferative cells (S+G2/M phases) substantially declined from 25.72 to 7.12%.Acidic culture resulted in a time dependent increase in the percentage of senescence cells,reaching a striking ~80% of cells after 5 days.NP-MSC increased levels of secreted IL-6 and IL-8 by NP-MSCs under pH 6.6 culture conditions." p53//p21//p27//Rb1//p16//MMP3//MMP9 Upregulation//Upregulation//Upregulation//Upregulation//Upregulation//Upregulation//Upregulation Western blot "Exposure of isolated NP-MSCs to acidic growth conditions resulted in relatively increased levels of p53,p21,p27,Rb1 and p16 proteins.MMP3 and MMP9 levels also increased whereas aggrecan." -- -- -- -- Human L cellular senescence 33824228 Sen_G_1339 LINC-PINT 378805 ncRNA "HCC,SPHC,SMMC-7721,MHCC-97H" -- Hepatocellular carcinoma Accelerate IHC//Western blot//DNA FISH//Flow cytometry//SA-β-gal activity Our results confirmed that Ki67 was remarkably lower in the PINT87aa overexpression group.CircPINT less expressed in proliferating than senescent SMMC-7721 and MHCC-97H cells.CircPINT expression in proliferating and senescent SMMC-7721 and MHCC-97H cells.The proportion of G1 phase cells was increased in PINT87aa-overexpressing cells.SA-β-gal staining showed a higher proportion of cellular senescence in HCC tissues with increased PINT87aa expression. FOXM1//PHB2 Downregulation//Downregulation Co-IP//immunfluorescence experiments PINT87aa could bind to the DNA-binding domain of FOXM.We observed co-localization of PINT87aa and FOXM1. -- -- -- -- Human L cellular senescence 33754036 Sen_G_1340 STING1 340061 protein coding Chondrocytes -- Osteoarthritis Accelerate SA-β-gal activity assay//Western blot "The results showed higher p16INK4a and p21 protein mea- sures in the STING-overexpressed and IL-1β-treated chondrocytes. Although STING knockdown significantly reduced the IL-1β-simulated upregulation of p16INK4a and p21 proteins, the knockdown of STING alone did not affect these proteins expression levels. We established that si-P65 remarkably reduced the expression of STING-induced senescence-associated (p16INK4a and p21), and apoptosis-associated (BAX, Cyto-c, as well as cleaved- caspase-3) proteins and increased the expression of BCL-2. The SA-β-gal and TUNEL staining confirmed that STING regulates senescence and apopto- sis via the NF-κB-signaling axis activation." -- -- -- -- NF-κB Activation Western blot//Immunofluorescence//SA-β-gal activity assay//TUNEL assay "The results showed that higher phosphorylation of P65 and iκb protein levels were seen in STING overexpressing and IL-1β treated chondrocytes.The analysis of the immunofluorescence of nuclear P65 confirmed that STING activates NF-κB signaling.STING mediates the degradation of the ECM degradation through the NF-κBsignaling cascade.STING regulates senescence and apoptosis via the NF-κB-signaling axis activation.Si-P65 remarkably reduced the expression of STING-induced senescence-associated (p16INK4a and p21),and apoptosis-associated (BAX,Cyto-c,as well as cleavedcaspase-3) proteins and increased the expression of BCL-2. The SA-β-gal and TUNEL staining confirmed that STING regulates senescence and apoptosis via the NF-κB-signaling axis activation." Human L apoptosis 33414452 Sen_G_1341 EZH2 2146 protein coding Chondrocytes Human cartilage explants Osteoarthritis Accelerate ELISA//RT-PCR//Safranin-O "We show that EPZ-6438 attenuated IL-1β-induced expression and release of MMP-1, -3 and -13 in chondrocytes, suggesting that this inhibitor could reduce chondrocyte catabolism. We confirmed this hypothesis using human cartilage explants. Safranin-O staining in explants treated with IL-1β for 48 h was less intense compared to untreated explants, showing that IL-1β induced a proteoglycan loss in cartilage. In the explants with the co-treatments of IL-1β with EPZ-6438, the safranin-O staining stayed intensively red, demonstrating that EPZ-6438 was able to preserve the proteoglycan content in the cartilage matrix, and consequently reduced cartilage degradation." NGF Upregulation RT-PCR "We showed that EZH2 overexpression increases IL-1β-mediated NGF expression. We showed that EZH2 overexpression increases IL-1β-mediated NGF expression, while EPZ-6438 had the opposite effect and reduced IL-1β-induced expression of NGF." -- -- -- -- Human L loss of proteostasis 33177650 Sen_G_1342 MIR126 406913 ncRNA DPSCs -- Aging Accelerate MTT assay "For control group, data displayed that there was no any influence, while miR‐126 caused a reduced cell proliferative rate.For DPSCs at PD54, miR‐126 inhibition significantly increased the cell replication of DPSCs during 72 hours post‐transfection." PTEN Downregulation qRT-PCR//Western blot "Here, the downregulation of PTEN was found in DPSC at PD54, compared with the DPSCs at PD16. Moreover, PTEN mRNA and protein level were also increased in senescent DPSCs transfected with miR‐126 inhibitor." Akt Activation Western blot "The downstream Akt phosphorylation was remarkably upregulated in DPSCs at PD54, while transfection with miR‐126 inhibitor caused an impaired Akt phosphorylation in DPSCs." Human HL apoptosis 33150661 Sen_G_1343 NFKBIA 4792 protein coding HDFs -- Aging Prevent RNA-seq//GO analysis The transcriptome of non‐irradiated NFKBIA‐depleted cells shared a strong overlap and the same GO terms with the senescent cells of the second phase.IκBα rescue through ectopic overexpression of an NFKBIA mutant encoding the proteasome‐insensitive IκBαS32AS36A blocked the induction of the bona fide SASP factors. -- -- -- -- -- -- -- -- Human HL apoptosis 33459422 Sen_G_1344 EZH2 2146 protein coding "HSCs,JS1,LX-2 " -- Aging Prevent Western blot//Flow cytometry//SA-β-gal activity assay//RT-qPCR//GO analysis "Treatment of primary HSCs with DZNep, or transient transfection with potent siRNAs for Ezh2 silencing, resulted in more quiescent HSCs-phenotype and pronounced growth retardation, remarkably weakened H3K27me2/3, and significantly downregulated α-SMA and COL1A.EZH2 inhibition by DZNep in mouse JS1 cells resulted in cell cycle arrest in S and G2 phases, lowered cell viability, increased cell senescence (in dose dependent mode), and higher percent of early apoptotic cells.DZNep treatment in rat primary HSCs, JS1 and LX-2 cells significantly lowered the transcriptional expression of cell proliferation marker protein Ki-67 coding gene MKI67 (marker of proliferation Ki-67).Key signaling pathways involved in cell cycle, DNA replication, mitotic cell cycle and spindle organization, and cytoplasmic ribosomal proteins were significantly inactivated. Some positive regulators of cell cycle including cyclins, cyclin-dependent kinases and E2F transcription factors, and of mitosis were pervasively downregulated; while the key negative regulators of cell cycle, Cdkn1a (cyclin dependent kinase inhibitor 1A), Gadd45a (growth arrest and DNA damage inducible alpha) and Gadd45b, the target genes of p53 and primary inhibitors of G2/M transition) were upregulated.(不知道用了什么实验)" -- -- -- -- TGF-β-SMAD Downregulation RNA-seq "154 out of the 351 associated DEGs in knowledgebase have measurement direction consistent with Tgfβ1 inhibition (P = 9.96E-30, Z-score = -2.087), the majority (127) of which are downregulated activator or effector genes, while a few (27) are upregulated inhibitor genes." Human HL apoptosis 33391480 Sen_G_1345 KDM6B 23135 protein coding "HSCs,JS1 " -- Aging Accelerate Western blot//ChIP-qPCR//RT-qPCR "Treatment of primary HSCs with GSK-J4, an jumonji H3K27me3 demethylase inhibitor of JMJD3 30, resulted in enhanced cell growth, morphological transition from quiescent HSCs to myofibroblast, reinforced H3K27me3, and upregulated fibrotic markers.DZNep-mediated EZH2 inhibition and adenovirus-mediated Jmjd3 overexpression in rat primary HSCs both consistently decreased H3K27me3 enrichment at promoters and gene bodies of Bambi, Cdkn1a, Gadd45a and Gadd45b, increased their transcriptional expression both at early (3 days) and late (6 days) stage of HSCs activation.We also observed substantial increase in protein expression for BAMBI, CDKN1A and GADD45B in HSCs with DZNep treatment or with overexpression of wild-type but not mutant Jmjd3." -- -- -- -- -- -- -- -- Human HL apoptosis 33391480