| Gene name: | CUL4B |
| Aging type: | Prevent |
| Aging characteristic: |
| Tissue type: | -- |
| Cell name: | NHF,U2OS |
| Experiment: | Knockdown//SA-β-gal activity assay//Western blot//BrdU assay |
| Description: | 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. |
| Regulatory pathway: | P53-ROS |
| R-AG-Pathway: | Downregulation |
| Official symbol(s): | TP53-ROS1 |
| Pathway experiment: | Knockdown//Flow cytometry//qRT-PCR//BrdU assay//SA-β-gal activity assay//Western blot |
| Pathway description: | 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 . |
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