Fig. 2: HBx enhances PTPN13 promoter methylation to decrease PTPN13 expression.

A Upper: CpG sites and CpG islands were identified by promoter sequence analysis. Lower: Five pairs of ChIP-qPCR primers were designed around the two CpG islands. B The promoter methylation levels of PTPN13 in the TCGA cohort were analyzed. C Correlations between PTPN13 expression and promoter methylation levels in 204 HCC tissue samples in the TCGA cohort were determined and then analyzed by Pearson’s correlation analysis. D The PTPN13 promoter methylation levels in HBV-negative HCC tissue samples (n = 3) and HBV-positive HCC samples (n = 4) were measured by Sequenom MassARRAY quantitative methylation analysis. E ChIP assays were performed in HBx stable expression and control cell lines using antibodies against DNMT3A; immunoprecipitated DNA was analyzed by qRT-PCR using primers described in (A) for amplifying the DNMT3A-binding regions in the PTPN13 gene promoter. F Prepared nuclear extracts were incubated with a biotinylated oligonucleotide probe corresponding to the FR9 region in binding site 3 in the PTPN13 gene promoter to perform an EMSA. Different fold excesses of unlabeled oligonucleotide probes for binding site 3 were used to compete with the interaction between the labeled probe and DNMT3A. The data represent the mean ± SD of three independent experiments (***P < 0.001 compared with the respective control).