| Gene name: | SAE2 |
| Aging type: | Prevent |
| Aging characteristic: |
| Tissue type: | -- |
| Cell name: | HCT116,U2OS |
| Experiment: | Knockdown//SA-β-gal activity assay//Flow cytometry//PI staining//BrdU assay |
| Description: | 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. |
| Regulatory pathway: | SUMO |
| R-AG-Pathway: | Activation |
| Official symbol(s): | SUMO |
| Pathway experiment: | SA-β-gal activity assay//Flow cytometry//DAPI staining//Western blot |
| Pathway description: | 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. |
Annotation:
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