Fig. 3.
Effects of HNF1 binding to the intron 1 ECR on the promoter activity of the OCT1 gene. Luciferase reporter gene assay were performed in the model hepatocellular carcinoma cell lines HepG2 (A and C) and Huh7 (B and D). The luciferase gene was cloned under the control of the Simian virus 40 promoter (PSV40) (A and B) or the 1.6-kb promoter fragment of the OCT1 gene (C and D). The constructs included an intact or mutated intron 1 ECR, or no ECR, cloned downstream of the luciferase gene. The coordinates are given in base pairs related to the distance to the transcriptional start site of OCT1. In HepG2 cells, the endogenous HNF1A expression was additionally downregulated using siRNA (white bars) (A and C). In Huh7 cells, HNF1A was additionally overexpressed by cotransfecting the overexpression plasmid pcDNA3::HNF1A with the reporter gene constructs (gray bars) (B and D). Shown are means and standard deviations of at least four independent experiments. (E) Effective downregulation and the overexpression of HNF1A were confirmed at the mRNA level by RT-qPCR (upper part) and at the protein level by Western blot (lower part). The RT-qPCR data represent mean and standard deviation of three independent experiments. The Western blot picture is a single representative experiment.