4OHT induces the expression of ERα in MDA-MB-231 cells. MDA-MB-231 cells were treated for 3 days with vehicle (control), 1 μM 4OHT or a combination of 1 μM 4OHT and 10 μM U0126 (4OHT/U0126). (A) ERα protein expression (red) in MDA-MB-231 cells was assayed by confocal microscopy. The nuclei were counterstained with DAPI (blue), and the merged images are shown. (B) ERα protein expression in MDA-MB-231 cells was assayed by western blot analysis. (C) Semiquantitative determination of ERα mRNA in breast cancer cells. The histograms were obtained by real-time PCR and represent the number of copies of mRNA for every 1 × 106 copies of β-actin. (D) The In vivo binding of ERα protein to the ERα promoter as determined by ChIP assays. Immunoprecipitation using anti-ERα or an IgG was performed in triplicate. The results are presented as the percentage of the input DNA. In this figure: *indicates that P < 0.05 when compared with untreated controls, and **indicates that the differences were significant (P < 0.05) when compared to 4OHT-treated cells but non-significant when compared to untreated controls.