Fig. 5

RGS2 regulates CDH1 promoter methylation. A The putative CpG sites within CDH1 promoter are shown. B Methylation status of 11 CpG sites on the promoter region of CDH1 gene. Methylation analysis was performed in ten clones for each group. Each row of circles represents a single clone, and each circle represents a single CpG site. Open circle represents unmethylated cytosine; filled circle represents methylated cytosine. C Western blot analysis for ALKBH1 expression levels in Flag-RGS2-transfected or control vector-transfected (Flag-RGS2, -) HTR-8/SVneo cells. D Western blot analysis for ALKBH1 expression levels in RGS2 siRNA-transfected (siRGS2) or control siRNA-transfected (siRGS2, -) HTR-8/SVneo cells. E Luciferase assay of HTR-8/SVneo cells co-transfected with firefly luciferase constructs containing the promoter construct of CDH1 and ALKBH1 or empty vector (Con) or treatment with decitabine for 24 h. F qRT-PCR analysis for E-Cadherin in HTR-8/SVneo cells transfected with ALKBH1 or treated with decitabine for 24 h. G Luciferase assay of HTR-8/SVneo cells co-transfected with firefly luciferase constructs containing the promoter construct of CDH1 and RGS2 and/or ALKBH1. H Luciferase assay of HTR-8/SVneo cells co-transfected with firefly luciferase constructs containing the promoter construct of CDH1 and miR-3935 mimics and/or TRAF6. I Luciferase assay of HTR-8/SVneo cells co-transfected with firefly luciferase constructs containing the promoter construct of CDH1 and miR-3935 inhibitor or negative control (NC). **p < 0.01 versus Con, vehicle, (RGS2 -, ALKBH1 -), mimics negative control (NC) without TRAF6 or inhibitor negative control (NC); ##p < 0.01 versus RGS2 without ALKBH1 or miR-3935 mimics with TRAF6; data represent the mean ± SD of three independent experiments