The expression of the promoter-hypermethylated genes was regulated epigenetically in glioma. (A) The expression of the promoter-hypermethylated genes in primary gliomas (n = 5) and non-tumor brain tissue samples (n = 5) was detected by quantitative RT-PCR. Data shown are mean value of individuals for each gene from three independent experiments. (B) The expression of the promoter hypermethylated genes in the 5-aza-2'-deoxycytidine-treated glioma cells. Data are expressed as mean% ± SEM of each group of cells for the indicated gene from three separate experiments. *p< 0.05 vs. control. (C) Correlation analysis of the levels of gene promoter methylation with its mRNA expression. The expression was analyzed with real time PCR, while the contents of promoter methylation in primary glioma samples were determined massarrays (n = 22-31 per group). Data shown are mean value of individual samples. (D) CHIP-PCR analysis for histone marks H3Ac (histone H3 acetylation), H3K4me3 (trimethyl-histone H3 lys4), and H3K9me3(trimethyl-histone H3 lys9) in the promoter-hypermethylated genes. U251, SF767, and SF126 cells were untreated or treated with 5-aza-2'-deoxycytidine. Input represents amplification from 1% chromatin before immunoprecipitation. IgG represents negative controls immunoprecipitated by normal rabbit serum. H3Ac, immunoprecipitated by rabbit anti-acytyl-H3; H3K4me3, immunoprecipitated by rabbit anti-trimethyl-H3(lys4); H3K9me3, immunoprecipitated by rabbit anti-trimethyl-H3(lys9).