Figure 4
RhoB downregulation effect on estrogen receptor alpha-dependent transcription and recruitment of cofactors on PR promoter. MELN or MCF-7 cells were transfected with siControl (siC), siB1 or siB2, and 3 days later were deprived of estradiol (E2) for 3 additional days. (A) MELN cells were then treated for 16 hours with E2 or ethanol. Luciferase activity was quantified and normalized. Error bars represent the mean values ± standard deviation (SD) from four independent experiments. Data generated in the absence of E2 are enlarged in the upper-right corner. (B) MCF-7 cells were then treated for 16 hours with E2 or ethanol and expression of the PR gene was measured. Error bars represent the mean values ± SD from three independent experiments. (C) MCF-7 cells were then treated for 16 hours with E2 or ethanol and progesterone receptor (PR) expression was analyzed by immunocytochemistry. ImmunoReactive Score (IRS) shown in the upper-right corner. Representative, respectively, of four and three independent experiments. (D) MCF-7 cells were treated for 1 hour with E2 and lysed for chromatin immunoprecipitation (ChIp) experiments using indicated antibodies. Specificity of the immunoprecipitation was controlled using nonspecific rabbit IgG. Recruitment of these proteins on the PR promoter was quantified. The fold induction for the recruitment of siB1 versus siC conditions was calculated. Error bars represent the mean values ± SD from three independent experiments. Differences were considered statistically significant at *P < 0.01 and **P < 0.001, Student's t test.