Fig. 3.

Evaluation of the RBL2 promoter activity, its methylation status and DNMT1 and DNMT3b mRNA expression in HuH-7 parental and HuH-7-CORE cell lines. a The pGL2bRb2 construct, containing the full-length promoter region of RBL2 linked to a luciferase reporter gene, was constructed and then transfected into HuH-7 and HuH-7-CORE cell lines. Measure of Luciferase activity (Luc/Renilla ratio) showed a more than three-fold down-modulation of RBL2 promoter activity in HuH-7-CORE cells. The values plotted are the mean of three independent transfections, for each of which two different aliquots have been analyzed for Luciferase quantitation. Statistical significance: *P = <0.05. b Methylation-specific PCR (MSP) analysis covering the region abundant in CpG sequences was carried out on genomic DNA from HuH-7 and HuH-7-CORE cells. U = Unmethylated; M = Methylated. c qPCR determination of DNMT1 and DNMT3b mRNA expression shows its up-regulation in HuH-7-CORE cells with respect to the parental HuH-7 cell line, whose value has been normalized to 1. The results shown are the mean of two independent experiments performed in triplicate. Statistical significance: *P = <0.05