Figure 3

Knockdown of E2F2 decreased cell growth in estrogen receptor-positive MCF-7 and T-47D cells but not estrogen receptor-negative MDA-MB-231 cells. (A) E2F2 mRNA levels decreased in MCF-7, T-47D and MDA-MB-231 cells following GW3965 treatment. (B) E2F2 mRNA levels decreased following transfection with targeting siRNA. (C) Protein expression of E2F2 following siRNA transfection at 72 hours post-transfection. (D) Trypan blue staining shows a decrease in MCF-7 and T-47D, but not MDA-MB-231 cell numbers following transfection with siE2F2 compared to control siRNA. (E) Relative mRNA expression of predicted E2F target genes after E2F2 siRNA transfection in MCF-7 cells. (F) Relative mRNA expression of predicted E2F target genes after E2F2 siRNA transfection in MDA-MB-231 cells. (G) E2F2 binding to promoter regions of CDC25A, EZH2, and CASP3 in MCF-7 cells was analyzed by chromatin immunoprecipitation assays. Disruption of E2F2 binding was observed following 24 hours of liver × receptor ligand treatment (filled bars) compared to vehicle-treated controls (open bars). No enrichment of binding or disruption following ligand treatment was observed in control immunoprecipitations using pre-immune immunoglobulin or in negative control regions in the exons of target genes. Significance was determined by Student's t-test (*P < 0.05, **P < 0.025, ***P < 0.01) and the error bars represent standard error of means from three replicate experiments.