Fig. 2

HBV induced hypermethylation of AP1 binding site in NFAT5 promoter for suppressing its expression. a Diagrams of reporter constructs containing the luciferase (Luc) gene under the control of the human NFAT5 promoter. L02 and Huh7 cell lines were co-transfected with pBlue-HBV or control plasmid pBlue-SK along with the constructed reporter plasmids with NFAT5 promoters. b NFAT5 promoter core region was identified in hepatoma cells by luciferase reporter gene assays. Length of 50 bp expresses a significant decrease of luciferase activity. *P < 0.05 (c) AP1 Binding Elements was identified by specific mutation. Mutant AP1 binding site represses luciferase activity. d Left panel: Analyses the binding of AP1 to the NFAT5 promoter by EMSA. AP1 probe was generated by annealing single-stranded and end-labeled oligo nucleotides containing the cognate NFAT5 promoter region. 1st lane: negative control reaction (labeled probe). 2nd lane: conventional reaction (including the nucleoprotein of activated target transcription factors with labeled probes). 3rd lane: probe cold competing reaction (including the nucleoprotein of activated target transcription factors, labeled probe and unlabeled probe with 100 times amount of labeled probe). 4th lane: cold competing reaction of mutated probe (including the nucleoprotein of activated target transcription factors, the labeled probe and mutated probe with 100 times amount of labeled probe). 5th lane: Super-shift reaction (including the nucleoprotein of activated target transcription factors, labeled probe and the specific antibody of AP1). Right panel: Determination the binding of AP1 to the NFAT5 promoter by ChIP assays. The lanes from left to right are Marker, experimental group, positive control group, input group, and negative control group. (E) Left panel: The methylation status of CpG island of NFAT5 promoter in response to HBV regulation was analyzed by bisulfite sequencing analysis in the Huh7 cells. Right panel: The methylation status of CpG island of NFAT5 promoter in response to HBV regulation was analyzed by MSP analysis as well