Supplementary Figure 2: BRG knockdown does not affect sensitivity to HDAC6 inhibition.

(a,b) Expression of HDAC6, FLAG and loading control β-actin in ARID1A-mutated OVISE cells with knocking down of endogenous HDAC6 expression using a shRNA that targets the 3’ UTR region of the human HDAC6 gene and concurrent expression of FLAG-tagged shRNA resistant wildtype HDAC6 or a catalytically inactivated H216/611A mutant (a). The indicated cells were subjected to colony formation assay and integrated density was measured with NIH Image J software as a surrogate for cell growth (b). n = 4 independent experiments. (c) ARID1A wildtype RMG1 cells with or without ARID1A knockdown were determined for Vorinostat dose responsive curves in a 12-day colony formation assay, n = 3 independent experiments. (d–f) ARID1A wildtype RMG1 cells with or without BRG1 knockdown were examined for expression of BRG1, HDAC6 or a loading control GAPDH by immunoblot (d); examined for HDAC6 mRNA expression by qRT-PCR (e), n = 3 independent experiments; or determined for ACY1215 dose responsive curves in a 12-day colony formation assay (f), n = 4 independent experiments. (g–i) BRM compensates for the knockdown of BRG1 at the HDAC6 gene promoter. ARID1A wildtype RMG1 cells were infected with the indicated shBRG1 or shControl. Expression of BRG1, BRM1, ARID1A and a loading control β-actin was determined by immunoblot (g). The indicated cells were subjected to ChIP analysis for the HDAC6 gene promoter using antibodies against BRG1 (h) or BRM (i). An isotype matched IgG was used as a control. n = 4 independent experiments. Error bars represent mean with S.E.M. P-value calculated via two-tailed t-test. Statistical source data are provided in Supplementary Table 6. Unprocessed original scans of all blots with size marker are shown in Supplementary Fig. 9.