Figure 1.
An shRNA screen identifies the SWI/SNF subunit ARID1B as a regulator of senescence. (A) Schematic model of the bypass of senescence screen using a library of shRNAs targeting genes deleted in HCC. An average of two different shRNAs per gene were used. MEFs were infected with the shRNA library or controls and passaged until vector cells reached senescence. shRNAs that extended life span were recovered by NGS. The screen was performed in triplicate. (B) List of significantly enriched shRNAs by Fisher's combined P-value (P < 0.001). shRNAs targeting ARID1B are marked in red. (C) Representative images of ARID1B, BrdU, SA-β-Gal immunofluorescence (IF) staining, and colony formation assay (crystal violet) of MEFs transduced with shRNAs targeting ARID1B, p16INK4a, or empty vector. DAPI was used to visualize the nuclei. Bar, 30 μm. Quantification of IF by high-content analysis (HCA) is shown at the right. (D) Soft agar assay of MEFs transduced with RasG12V cDNA and either an empty vector or vectors carrying shRNAs for p16/Arf or ARID1B. Representative images at 4× magnification (left) and quantification (right) are shown. Graphs in C and D represent mean ± SD from n = 4 and n = 3, respectively. (***) P < 0.001 by two-tailed Student t-test.