Expression of Bmi1 was stimulated by E2 in ERα-positive breast cancer cells. (A) Effect of Bmi1 ectopic expression on ERα protein in MCF-7 cells. MCF-7 cells were transiently transfected with Bmi1 (pcD-Bmi1), an empty vector, or transfection reagent (control). Cells were collected after 48 h of transfection and analyzed for Bmi1, ERα and β-actin expression with Western blot. This image represents one of three experiments. (B-D) Expression of Bmi1 was stimulated by E2 in ERα-positive breast cancer cells. ERα-positive MCF-7 (B) and ERα-negative MDA-MB-231 (C) cell lines were cultured in phenol red free medium containing 10% charcoal striped FBS for 72 h and 10−8 M E2 was added. At indicated time points, cells were collected and analyzed for Bmi1, ERα, p16INK4a and β-actin expression by Western blot and real time RT-PCR (B’, right panel). (D) MCF-7 cells were treated with 10−6 M OHT in the presence of E2 and Western blot was performed. β-actin was used as loading control. Quantitative analyses of ERα, Bmi1 and p16INK4a are presented. All data were obtained from three independent experiments and are shown by bars as means ± SD (*,# or △P < 0.05, **,## or △△P < 0.01, ***,### or △△△P < 0.001 when ERα, Bmi1 and p16INK4a were compared with the control group, respectively).