Figure 5.
A focused shRNA screen identified novel ARID1B effectors controlling senescence. (A) Schematic model of the bypass of senescence screen using a library of shRNAs targeting genes up-regulated in OIS, including some that are ARID1B-dependent. An average of six different shRNAs per gene were used. After infection with the shRNA library or controls, IMR90 ER:RAS cells were treated with 4OHT and passaged until library-infected cells bypassed senescence. shRNAs that bypassed senescence were recovered by NGS. The screen was performed in duplicate. (B) List of significantly enriched genes by Fisher's combined P-value (P < 0.001). CDKN1A and genes selected for further analysis are marked in red. (C–E) Colony formation assay (C), BrdU incorporation (D), and p21 analysis (E) of IMR90 cotransduced with RASG12V (or empty vector) and shRNAs for ENTPD7 (shE2 and shE3), SLC31A2 (shS1 and shS2), or NDST2 (shN1 and shN2). A p53 shRNA was used as control. Quantifications of IF staining were performed by HCA. n = 3. Graphs represents mean ± SD. (***) P < 0.001; (**) P < 0.005; (*) P < 0.05 by two-tailed Student t-test.