ANT2 knockdown-induced JNK signaling up-regulates DNMT1, leading to methylation of the DcR2 promoter in MCF7 cells. (A) Methylation-specific PCR analysis of effect of ANT2 shRNA on DcR2 genomic DNA of MCF7 cells. After cells were transfected with scrambled or ANT2 shRNA for 24 h, they were lysed, and the genomic DNA was purified. Unmethylated cytosines were converted to uracils using an Invitrogen MethylCode Bisulfite Conversion Kit, and the converted genomic DNA was used for PCR with methylation-specific primers. The cell extracts were also analyzed for ANT2 and β-actin expression by immunoblotting. (B) RT-PCR analysis of the effect of ANT2 shRNA-induced methylation on DcR2 expression. Cells were pre-treated with the methylation inhibitor 5-aza-dC for 2 h and then transfected with scrambled or ANT2 shRNA. (C) RT-PCR analysis of effect of ANT2 shRNA-induced JNK signaling on DNMT1 expression. MCF7 cells were pre-treated with SP600125 for 2 h and then transfected with scrambled or ANT2 shRNA. Cell extracts were also analyzed for DNMT1 and β-actin expression by immunoblotting. (D) Effect of ANT2 shRNA-induced JNK signaling on DNMT1 activity. MCF7 cells were treated as in (C), and nuclear extracts were evaluated for DNMT1 activity. (E) Effect of JNK inhibition on ANT2 shRNA-induced sensitization to TRAIL. MCF7 cells were treated with SP600125 and scrambled or ANT2 shRNA as in (C), and 24 h after transfection, they were treated with recombinant human TRAIL (100 ng/ml) for 12 h. Cell viability was measured using a CCK8 assay kit.