ER beta silencing counteracts the effects of mibolerone on p21, cyclin D1 expression and cell proliferation. (A) RNA was extracted from MCF-7 cells treated with vehicle (−) or Mib 10 nM for 48 hours in the presence of a non-specific or an ER beta siRNA, reverse transcribed and cDNA was subjected to Real-Time RT-PCR for p21 and cyclin D1 mRNA expression. Each sample was normalized to GAPDH mRNA content. Data represent the mean ± S.D. of values from three separate RNA samples expressed as the percentage of the control (−) assumed to be 100%. (B) Western blot analysis for p21 and cyclin D1 expression in cells treated with vehicle (−) or Mib for 48 hours in the presence of a non-specific or an ER beta siRNA. GAPDH was used as the loading control. The histograms represent the mean ± S.D. of three independent experiments in which band intensities were evaluated in terms of optical density arbitrary units and expressed as the percentage of the control assumed to be 100%. (C, D) MTT assays in MCF-7 and ZR-75 cells transfected and treated as indicated. Results are expressed as fold change ± S.D and are representative of three different experiments each performed in triplicate. *, P <0.01 compared to vehicle-treated cells. **, P <0.01 ER beta siRNA-transfected cells compared to Mib-treated cells. ER, estrogen receptor; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide.