Fig. 6

EZH2 represses ATOH8 by controlling the methylation of its promoter region. a, The scheme shows CpG island percentage in the promoter region of ATOH8. CpG islands were predicted using MethPrimer (http://www.urogene.org/methprimer/index.html). The regions analyzed by BSP are indicated. The sequence of the ATOH8 promoter region analyzed by BSP is shown. The CpG dinucleotides within this region are numbered as 1–23. b, The methylation status of the ATOH8 promoter region in Bel-7402, HepG2, SK-Hep-1, SNU-387 and MHCC-97H cell lines. The open and filled circles indicate the unmethylated and methylated CpGs, respectively. c, qRT-PCR analysis of ATOH8 expression in indicated cells treated with 5 mmol of 5-aza-dC for 72 h (*, P < 0.05). d, The graph shows the methylation status of the ATOH8 promoter in 12 pairs of HCC and peritumoral tissues. e, The graph shows the relative expression of ATOH8 by qRT-PCR in 12 pairs of HCC and peritumoral tissues. f, The methylation status of the ATOH8 promoter was examined by BSP in HepG2 and Bel-7402 cells after transfection of shEZH2 or shEZH2scr (n = 3, *P < 0.05). M: methylated, U: unmethylated. g, Protein expression of ATOH8, EZH2, H3K27me3, and Histone H3 by Western blot analysis in shEZH2 or shEZH2scr transfected HepG2 cells and empty or EZH2 transfected Bel-7402 cells. mRNA expression of ATOH8 was examined by RT-PCR in these cells. β-Actin was used as a loading control. h, RT-PCR analysis of EZH2 and H3K27me3 enrichment upstream of ATOH8 in shEZH2 or shEZH2scr transfected HepG2 cells and empty or EZH2 transfected Bel-7402 cells. i, ChIP–qPCR analysis of EZH2 and H3K27me3 enrichment upstream of ATOH8 in shEZH2 or shEZH2scr transfected HepG2 cells and empty or EZH2 transfected Bel-7402 cells (n = 3, *P < 0.05)