Fig. 5. Hypermethylation of transcriptional activator Sp1-binding site in the ZNF132 promoter region leading to ZNF132 gene silencing in esophageal cell line.

a Hypermethylation of the ZNF132 promoter region decreases the gene expression of ZNF132. Sp1 and ZNF132 promoter regions were transferred into HEK293T cells to determine activity. Data are presented as the mean ± SD. ***P < 0.001. b Methylated luciferase reporter assay shows that methylated Sp1-binding site resulted in a decrease in luciferase activity compared to the unmethylated site. Data are presented as the mean ± SD. *P < 0.05, ***P < 0.001. c ChIP assay to show that Sp1 protein can bind to the ZNF132 promoter region containing Sp1 site in in vitro cultured cells. d The protein molecular weights of Sp1 is 90 kDa. DNA pull-down assay to confirm the hypermethylated probe of this segment had significantly lower binding affinity to Sp1 than the unmethylated probe