Figure 4

EGCG and TSA alters the binding of transcriptional repressor complex to the ERα promoter. A) MDA-MB-231 cells were treated with EGCG and/or TSA as described previously and analyzed by ChIP assay together with untreated control cells. Chromatin DNA from MCF-7 and MDA-MB-231 cells was immunoprecipitated with antibodies against proteins of the ERα transcriptional repressor complex including HDAC1, SUV39H1, DNMT1, E2F-4, Rb/p130 and p300 together with mouse IgG control. MCF-7 cells served as a positive control. Inputs came from the total DNA and served as the same ChIP-PCR conditions. The purified ChIP-DNA was amplified by PCR with the use of ERα promoter primers. Enrichment values were quantified and normalized to the corresponding inputs while the untreated MDA-MB-231 control sample is represented as 1.0. Representative photograph shown from three independent experiments. B) ChIP data were calculated from the corresponding DNA fragments amplified by ChIP-PCR; columns, mean; bars, SD. C) The protein levels of epigenetic regulators, HDAC1, SUV39H1 and p300, were determined by western-blot analysis. MCF-7 and MDA-MB-231 cellular proteins were extracted after corresponding treatments. Protein lysates (50 μg) were resolved on 12% SDS-PAGE, transferred onto nitrocellulose membrane, and probed with antibodies against HDAC1, SUV39H1 and p300. Membranes were re-probed with anti-GAPDH antibody to ensure equal loading. Representative photograph shown from the experiments repeated in triplicate.