Mibolerone up-regulates ER beta expression in breast cancer cells. (A) 3H Thymidine incorporation assays in MCF-7 and ZR-75 cells treated with vehicle (−) or mibolerone (Mib) 10 nM for two, four and six days. Columns, mean of three independent experiments, each performed with triplicate samples expressed as percent of control. (B) Total RNA was isolated from MCF-7 and ZR75 cells treated with vehicle (−) or Mib 10 nM for 24 and 48 hours, and reverse transcribed. cDNA was subjected to real time PCR using specific primers for ER beta and GAPDH. Each sample was normalized to its GAPDH mRNA content. Data represent the mean ± S.D. of values from three separate RNA samples expressed as a percentage of control assumed to be 100%. (C) Bottom panel, Western blot analysis of ER beta expression in total protein extracts from MCF-7 and ZR75 cells, treated with vehicle (−) or Mib 10 nM for 24 and 48 hours. GAPDH was used as loading control. Upper panel, 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%. *, P <0.05 compared to vehicle-treated cells. ER, estrogen receptor.