Fig. 2

DNMT1-dependent DNA methylation inhibits STAT6 expression. a Distribution of CpG islands in the − 1000 to + 1400 STAT6 promoter region. Two underlined sequences show the locations of MSP-PCR primers; 14 analyzed CpG sites are shown in bold red font. b MSP of STAT6 promoter CpG islands in non-tumor brain tissue (NTT), low-grade glioma tissue (LGG), and high-grade glioma tissue (HGG). M, methylated product; U, unmethylated product. c Bisulfite sequencing of 14 CpG sites in STAT6 promoter CpG islands in normal human astrocytes (NHAs), NTT, brain metastatic adenocarcinoma (BMA), LGG, and HGG tissue. Each square represents 1 CpG analyzed, and colors indicate the percentage of methylation. d Immunoblot of STAT6 (left) and bisulfite sequencing (right) of STAT6 promoter CpG islands in the indicated GBM cell lines. e RT-qPCR of STAT6 (top) and immunoblot of indicated proteins (bottom) in U373MG cells treated with indicated doses of 5-Aza for 72 h. Results are presented as means ± SD (error bars) of three independent experiments (*p < 0.05 and **p < 0.01 vs. untreated cells). f Immunoblot of the indicated STATs and DNMTs in U373MG cells transfected with the indicated DNMT siRNA (DNMTsi) or control siRNA (CONsi). g, h U373MG cells transfected with CONsi or indicated doses of DNMT1si, followed by quantification of STAT6 mRNA by RT-qPCR (g) and immunoblotting for DNMT1 and STAT6 in cells from g (h). Results are presented as means ± SD (error bars) of three independent experiments (*p < 0.05 vs. CONsi-transfected cells). i RT-qPCR of STAT6 in LN229 cells treated with indicated doses of 5-Aza for 72 h (left) and immunoblot of STAT6 and the indicated DNMTs in LN229 cells transfected with the indicated DNMT siRNA or control siRNA (right). Results are presented as means ± SD (error bars) of three independent experiments (*p < 0.05 and **p < 0.01 vs. untreated cells)