Fig. 3

IL-32γ suppresses HBV transcription by downregulating viral enhancer/core promoter activities. a, b At 72 h post-co-transfection of Huh7 cells with HBV 1.2 and IL-32γ vectors, the levels of HBV RNAs and proteins were determined by Northern and western blotting, respectively. The 18S RNA was used as a loading control. c Cartoon of various HBV enhancer reporter mutants used in this study. d Effect of IL-32γ on each enhancer reporter mutant in Huh7 cells. Relative luciferase activity of each enhancer clone was determined at 48 h post-co-transfection with either empty or IL-32γ vector. e HBV 1.2 and siRNAs (20 nM) were co-transfected into Huh7 cells. Next day, the cells were treated with cytokines (TNF-α and IFN-γ) in fresh medium. At 72 h post-transfection, the cells were harvested and the levels of HBV RNAs were determined by Northern blot analysis. f Huh7 cells were co-transfected with HBV 1.2 and empty or IL-32γ vector. Next day, the cells were treated with IFN-γ. At 72 h post-transfection, the cells were harvested and nuclear and cytoplasmic fractions were isolated. Total RNA was extracted and subjected to Northern blot analysis. g Analysis of HBV transcription rate by nuclear run-on assay using the nuclear fractions shown in f. The level of HBV transcription was normalized to that of GAPDH RNA. Data (d) were obtained from three independent experiments (mean ± S.D.)