Mibolerone recruits AR protein to an ARE site in the ER beta gene promoter. (A) Nuclear extracts from MCF-7 cells treated with vehicle (−) or Mib 10 nM for 16 hours were incubated with a double-stranded ARE specific sequence probe labeled with [γ32P] ATP and subjected to electrophoresis in a 6% polyacrylamide gel (lanes 1 and 4). Competition experiments were performed adding as the competitor a 100-fold molar excess of unlabeled probe (lane 2 ) or a 100-fold molar excess of unlabeled oligonucleotide containing a mutated ARE motif (lane 3). Nuclear extracts from MCF-7 cells treated with Mib 10 nM for 16 hours were incubated with anti-AR (lane 5) or IgG (lane 6) antibodies, in the presence of the probe. Lane 7, probe alone. (B,C) , MCF-7 cells treated with vehicle (−) or Mib 10 nM for 16 hours were cross-linked with formaldehyde and lysed. The pre-cleared chromatin was immune-precipitated with specific anti-AR, anti-polymerase II antibodies, and with a normal mouse serum (IgG) as a negative control. For each sample and input, a 5 μl volume was analyzed by real time PCR with specific primers, as detailed in the Methods Section, to amplify the ER beta promoter sequence containing the ARE site. The histograms represent the mean ± S.D. of three independent experiments. *, P <0.05 compared to vehicle-treated cells; *, P <0.01 compared to Mib-treated cells. AR, androgen receptor; ARE, androgen response element; ER, estrogen receptor; IgG, immunoglobulin G.