Supplementary Figure 4: ARID1A regulates HDAC6 expression.

(a) Expression of HDAC6, BRG1 and a loading control β-actin in ARID1A-mutated TOV21G cells with or without wildtype ARID1A restoration and with or without concurrent BRG1 knockdown. ARID1A restoration the same as Fig. 3f. (b) Relative expression of the indicated HDACs mRNA determined by qRT-PCR in ARID1A-mutated TOV21G cells with wildtype ARID1A restoration or ARID1A wildtype RMG1 cells with ARID1A knockdown. n = 3 independent experiments. ^∗P < 0.01. P-value calculated via two-tailed t-test. (c) Tumour cells derived from the indicated genetic mouse endometrioid tumours were treated with ACY1215 (1.25 μM). The percentage of surviving cells was determined by colony formation. n = 3 independent experiments. (d) Relative mRNA expression of the indicated class II HDACs in ARID1A wildtype (n = 12) and mutated (n = 7) human ovarian clear cell carcinoma specimens. Note that none of them are significant. (e) ARID1A expression negatively correlates with HDAC6 expression at the mRNA level in clear cell and endometrioid ovarian cancer cells in the cell lines encyclopedia¹. Pearson correlation was used for calculating P value. (f) ARID1A expression negatively correlates with HDAC6 expression at the mRNA level in a published dataset of laser capture microdissected (LCM) clear cell ovarian carcinomas and normal human ovarian surface epithelial cells². Pearson correlation was used for calculating P value. (g) Schematic of primers’ positions for ChIP-PCR in the human HDAC6 gene locus. (h,i) ARID1A wildtype RMG1 cells with or without ARID1A knockdown were subjected to ChIP analysis using antibodies against ARID1A (h), anti-Pol II (i) or an IgG control. ChIP products were subjected to qPCR analysis using primers as indicated in (g) for the human HDAC6 gene locus. n = 4 independent experiments. (j) ARID1A wildtype RMG1 cells with or without ARID1A knockdown were subjected to ChIP analysis using an antibody against acetylated histone H3 (H3Ac) or an IgG control for the human HDAC6 gene locus. 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.