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. 2016 Oct 1;30(19):2187–2198. doi: 10.1101/gad.286112.116

Figure 3.

Figure 3.

ARID1B regulates OIS in human cells. (A) IMR90 ER:RAS cells were transduced with either an empty vector (vec), a p53 shRNA (shp53), or ARID1B shRNAs (shARID1B.2 [shA2] and shARID1B.3 [shA3]) and induced to undergo senescence by 4OHT treatment. Cell proliferation was assessed by crystal violet staining (top left) and BrdU incorporation (right). Senescence was measured by SA-β-Gal staining (representative pictures are at the bottom left, and quantification is at the right). (B, left) IMR90 ER:RAS cells transduced with an empty vector (±4OHT) or ARID1B shRNAs (+4OHT) were subjected to global mRNA expression analysis. Data were subjected to GSEA and IPA. n = 3 biological replicates were used. (Middle) GSEA showing loss of a signature associated with OIS in the gene expression profile of IMR90 ER:RAS shARID1B versus empty vector, both 4OHT-treated. (NES) Normalized enriched score; (FDR) false discovery rate. (Right) IPA upstream regulator analysis of the indicated comparisons is shown. The predicted activation (z-score) of SMARCA4, TP53, and reactive oxygen species (ROS) pathways is shown. (C,D) Quantification by HCA (D) of IF staining (C) for p21CIP1, p16INK4a, p53, γH2AX, and 8-oxodG in IMR90 ER:RAS cells transduced with either an empty vector or an shRNA targeting ARID1B (±4OHT). DAPI was used to visualize the nuclei. Graphs represent mean ± SD from n = 4 (A, BrdU) and n = 3 (A [SA-β-Gal], D). (***) P < 0.001; (**) P < 0.01; (*) P < 0.05 by two-tailed Student t-test. Bars, 30 μm.