Figure 2.

DNA methyltransferase 1 (DNMT1)-mediated upregulation of CLDN6 expression by SB431542. (A,B) Real-time polymerase chain reaction (RT-PCR) and immunoblot analysis of DNMT1 and CLDN6, and densitometric analysis of relative gene expression levels after normalization to loading controls GAPDH and β-actin are presented. (C) DNMT1 activity assays. (D) Methylation-specific PCR (MSP) analysis of CpG island of CLDN6 promoter using bisulfite-treated genomic DNA isolated from MCF-7 and SKBR-3 cells. (E) CpG island methylation within the CLDN6 promoter region was measured by bisulfite sequencing in SKBR-3 cells. “Me” stands for methylated, and “U” stands for unmethylated. (F) Chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) assay to detect the binding of DNMT1 to the promoter of CLDN6 (** p < 0.01). The lane “M” stands for marker; bars represent mean ± SE (n = 3).