Fig. 5
ZC3H18 promotes binding of E2F4 and activation of the BRCA1 promoter. a qRT-PCR analysis of BRCA1 mRNA expression. OVCAR-8 cells transfected with control luciferase (Luc), E2F2, E2F3, E2F4, E2F5, E2F6, E2F7, or E2F8 siRNAs with or without ZC3H18 siRNA were analyzed by qRT-PCR for BRCA1 mRNA levels, which were normalized to GAPDH mRNA. b Immunoblots of indicated proteins in OVCAR-8 and PEA1 cells transfected with control luciferase (Luc) and two independent E2F4 siRNAs. c BRCA1 mRNA expression, normalized to GAPDH mRNA, was determined by qRT-PCR in short-term ex vivo cultures of HGSOC tissues from PDX models electroporated with Luc or E2F4 siRNAs. d ZC3H18 promotes E2F4 occupancy on the BRCA1 promoter. OVCAR-8 cells transfected with control luciferase (Luc) or ZC3H18 siRNAs were analyzed by ChIP for E2F4 bound to the BRCA1 promoter. e, f Depletion of E2F4 or ZC3H18 promotes E2F1 and DNMT1 occupancy on the BRCA1 promoter. OVCAR-8 cells transfected with Luc, E2F4, and ZC3H18 siRNAs were analyzed by ChIP for E2F1 (e) and DNMT1 (f) accumulation on the BRCA1 promoter. g E2F4 depletion disrupts HR. OVCAR-8-DR-GFP cells transfected with pCβASceI plus indicated siRNAs were analyzed for GFP fluorescence by flow microfluorimitry 48 h after transfections. HR efficiencies were normalized to control (Luc) siRNA-transfected cells. h OVCAR-8 cells were transfected with control luciferase (Luc), E2F4, or BRCA1 siRNAs. Forty-eight hours later, the cells were trypsinized, re-plated, and allowed to adhere for 24 h. The indicated concentrations of olaparib were then added, and the cells were cultured for 10 days, stained with Coomassie Blue, and colonies were counted manually. Data are means ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired Student’s t-test. Representative immunoblots in b are provided from three independent experiments. Unprocessed blots are provided in Source data file. The graph in h represents one of three independent experiments that gave similar results. Error bars are standard deviation of triplicate wells from a representative experiment
