Fig. 4

ZEB1 stimulates DNMT1 transcription via increasing its promoter region histone acetylation. A Detection of ZEB1 expression in breast cancer cell lines and MCF10A mammary epithelial cell line. B, C Analysis of DNMT1 expression by western blot (B) and qRT-PCR (C) in MCF7-Vector cells and MCF7-ZEB1 cells. D, E Assessment of DNMT1 expression by western blot (D) and qRT-PCR (E) after silencing DNMT1 in MDA-MB-231 cells. F, G Detection of DNMT1 expression by western blot (F) and qRT-PCR (G) after treated with P300 specific inhibitor (60 nM) in MCF7-Vector cells and MCF7-ZEB1 cells. H–J ChIP assays on DNMT1 promoter were done using anti-ZEB1, anti-P300 and anti-Acetylation antibodies in MCF7-Vector and MCF7-ZEB1 cells. K–M Detection the enrichment of ZEB1, P300 and histones acetylation on DNMT1 promoter by ChIP assays in MDA-MB-231-shCtrl cells and MDA-MB-231-shZEB1 cells. N–Q Luciferase reporter assays detected the DNMT1 promoter region activity after ectopic expression of DNMT1 (K) or treated by 500 nM DAC (L) in HEK-293T cells; DNMT1 expression was detected by immunoblot assay in HEK-293T-Vector and HEK-293T-DNMT1 cells (J). Data are represented as mean ± SD of three independent experiments, and *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test)