Fig. 3

USF-1 is an important transcription factor that regulates C9 expression and is inhibited by HBX. a Relative luciferase activity of C9 promoter in Huh7 cells transfected with pcDNA3.1/myc-His(B)-HBx, pGL3-C9-Prom and pRL-CMV Renilla Luciferase Reporter vector along with different concentrations (0, 400, 800 and 1200 ng/ml) of pCMV-USF1 plasmid. b Relative C9 mRNA expression following transfection of Huh7 cells with pcDNA3.1/myc-His(B)-HBx and pCMV-USF1 plasmid in different concentrations (0, 400, 800 and 1200 ng/ml). c Relative C9 promoter luciferase activity in Huh 7 cells transfected with pGL3-C9-Prom, pRL-CMV Renilla Luciferase Reporter vector and different concentrations of antisense oligonucleotide designed against USF-1 gene (USF-1_ASO) (0–1200 ng/ml). d Relative C9 mRNA expression following transfection of Huh7 cells with USF-1_ASO (1000 ng/ml) or scrambled oligo control (ASO_Ctrl) (1000 ng/ml). e Assessment of relative C9 luciferase activity upon transfection of Huh7 cells with wild-type pGL3-C9-Prom and its mutated derivative pGL3-C9-Prom-mt in presence or absence of USF-1 expressing plasmid (pCMV-USF1). f ChIP analysis of USF1 binding to C9 promoter in HBx and empty vector transfected Huh7 cells. ChIP analysis was performed using IgG (negative control) and anti-USF-1 antibody. The relative enrichment of C9 promoter DNA was normalized to the input DNA (5%) for each experiment. Mean ± SD are based on three independent experiments. Paired t tests were performed for comparing paired groups in d, f. Comparisons between groups were performed using the one-way ANOVA with p values adjusted by the Tukey’s multiple comparison test in e. **p < 0.005, ***p < 0.0001