Expression of Bmi1 was stimulated by E2 in ERα-restored breast cancer cells. (A) 231/ERα and 231/vec were generated by stable transfection of MDA-MB-231 cells by ERα or empty vector, respectively. (B) 231/ERα and (D) 231/vec cells were stimulated with 10−8 M E2, and 10−6 M OHT was added at the same time, (C and E). At indicated time, cells were collected and analyzed for Bmi1, ERα and β-actin expression by Western blot and real time RT-PCR (B’, right panel). 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 (#P < 0.05 when Bmi1 was compared with the control group).