Figure 3.

E2 Induces ERα, STAT5b, and Phosphorylated RNA Polymerase II Recruitment to the Cyclin D1 and c-Myc Promoters. STAT5b Knockdown Inhibits E2-induced Recruitment of ERα and Phosphorylated RNA Polymerase II. A-B, T47D cells were nucleofected with 2.5 μg of 20 μm control or STAT5b siGENOME SMARTpool siRNA (Dharmacon) per reaction, grown in E2-free conditions for three days, then treated with control media containing ethanol (V) or 10 nm E2 for 30 min. ChIP assays were performed using either no antibody (-Ab) or antibodies directed against ERα, STAT5b, and the phosphorylated RNA Pol II CTD. The immunoprecipitated DNA was quantitated by real-time PCR with the use of primer sets directed towards the E2-responsive regions of the cyclin D1 (A) and c-Myc (B) promoters. The data represent the mean ± SEM of four experiments done in triplicate. Two-way ANOVA was used to determine statistical significance for –Ab, ERα, and STAT5b. *, P < 0.05 vs. V; #, P < 0.01 vs. E2-stimulated siCon cells. Student’s t test was used to determine statistical significance for ph-Pol II. *, P < 0.05 vs. V; #, P < 0.05 vs. E2-stimulated siCon cells. Inset, Western blot verifying STAT5b knockdown prior to 30 min E2 treatment. T47D cells were nucleofected with 2.5 μg of 20 μm control or STAT5b siRNA and lysed after 72 h. Proteins were separated by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies specific for the STAT5 SH2 domain or β-Actin.