Figure 2

Epigenetic alterations in response to GE and/or TSA treatments. (A) Histone modification patterns in the ERα promoter were analyzed by ChIP assay. Representative photograph from an experiment was repeated in triplicate. (B) Histone modification enrichment in the ERα promoter was calculated from the corresponding DNA fragments amplified by ChIP-PCR as shown above. MDA-MB- 231 cells were treated as described previously and analyzed by ChIP assays using chromatin markers including acetyl-H3, acetyl-H3K9, acetyl-H4, dimethyl-H3K4 and mouse IgG control in the promoter region of ERα. Inputs came from the total DNA and served as the same ChIP-PCR conditions. DNA enrichment was calculated as the ratio of each bound sample divided by the input while the untreated MDA-MB-231 control sample is represented as 1. (C) HDACs enzymatic activity. (D) DNMTs enzymatic activity. Nuclear proteins of MDA-MB-231 cells were extracted after the treatment as described above. The HDACs and DNMTs activity assays were performed according to the manufacturer’s protocols. (E) Binding abilities of HDACs and DNMTs in the ERα promoter were determined by ChIP assay as described previously. The values of enzymatic activities of HDACs and DNMTs are the means of three independent experiments. Columns, mean; Bars, SD. *, P < 0.05, significantly different from control; £, P < 0.05, significantly different from GE; †, P < 0.05, significantly different from TSA. (F) The protein level changes of HDACs and DNMTs were determined by western-blot analysis. GAPDH antibody was used to ensure equal loading. Representative photograph from an experiment was repeated three times.