Fig. 2

Promoter methylation and histone H3 deacetylation were involved in GPER down regulation in CRC cell and tissues. The mRNA (a) and protein (b) levels of GPER in CRC cell lines, human colon mucosal epithelial NCM460 cells were measured by qRT-PCR and western blot analysis, respectively; (c) Methylation status of GPER promoter in CRC cell lines was determined by bisulfite genomic DNA sequencing. Each dot represents a CpG site. White dots represent unmethylated CpG dinucleotides whereas each black dots represents a methylated cytosine residue within the CpG islands; (d) Methylation statuses of GPER promoter in five pairs of CRCs tissues and patient-matched normal tissues (Cohort 1) were determined by bisulfite genomic DNA sequencing; (e) HCT-116 or SW480 cells were treated with 5 μM 5-Aza for the different times, then mRNA of GPER was measured by use of qRT-PCR; (f) ChIP analysis of NCM460 and CRC cell lines were conducted on the GPER promoter regions by use of antiacetyl histone H3 antibody; (g) The correlation between relative acet-H3 enrichment and GPER mRNA expression in LS147T, HCT-116, SW620, HCT8, and SW480 cells; (h) ChIP analysis of five pairs of CRCs tissues and patient-matched normal tissues (Cohort 1) were conducted on the GPER promoter regions by use of antiacetyl histone H3 antibody. Data were presented as means ± SD of three independent experiments. *p < 0.05 compared with control; **p < 0.01 compared with control