Fig. 2

The selectivity against CARM1 by EZH2 inhibition correlates with apoptosis induction in a methyltransferase activity-dependent manner. a Relative growth of the indicated HOSE, FTE, and EOC cancer cell lines with high or low CARM1 expression treated with 10 μM GSK126 or vehicle in a colony formation assay as determined by NIH ImageJ quantification. b Expression of H3K27Me3 in the indicated EOC cell lines with high or low CARM1 expression treated with or without 10 μM GSK126. Expression of β-actin was used as a loading control. c, d Expression of CARM1, BAF155Me, and β-actin in OVCAR3 cells with ectopic CARM1 or control (c). GSK126 dose response curves of the indicated cells were determined by colony formation assay (d). Mean of three independent experiments with SEM. e Control parental and CARM1 knockout A1847 cells were treated with 10 μM GSK126 or vehicle DMSO control for 7 days. Percentage of Annexin V-positive apoptotic cells was quantified. *P < 0.001 and ^#P > 0.05. f Apoptosis markers cleaved caspase 3 and cleaved PARP p85 in parental and CARM1 knockout A1847 cells treated with 10 μM GSK126 for 7 days. Expression of β-actin was used as a loading control. g–j CARM1-high A1847 cells were infected with a lentivirus encoding shEZH2 targeting the 3′ untranslated region (UTR) of the human EZH2 gene together with a retrovirus encoding wild-type EZH2 (WT) or a SET domain-deleted EZH2 mutant (EZH2 ΔSET). Drug-selected cells were examined for EZH2, H3K27Me3, apoptosis markers cleaved caspase 3 and cleaved PARP p85, and a loading control (β-actin) by immunoblot (g), quantified for Annexin V-positive apoptotic cells by FACS (h), subjected to a colony formation assay (i), and quantified for the relative cell growth based on colony formation using NIH ImageJ (j). *P < 0.01. Means of three independent experiments with SEM. P-values are from two-tailed t-test