Figure 2.

Effect of HBx on HIF expression and transcriptional activity in HepaRG cells. HepaRG cells encoding HBx were incubated with Tet (50 µM) for 24 h and HBx protein and Smc6 expression detected by western blot, uncropped blots are available in Supplementary Fig. 2 (a). Differentiated HepaRG cells encoding WT or mutated HBx (STOP) were treated or not with tetracycline (1 µM) and infected with HBV_WT or HBV_X- and 13 days later infection assessed by measuring total viral RNAs. The data is normalised for each cell line relative to HBV_WT infection without Tet and represent the mean of 3 independent experiments; 2-way-ANOVA with Bonferroni correction was applied with p < 0.05 deemed as significant (b). HepaRG cells encoding WT or mutated HBx (STOP) were incubated with or without Tet (50 μM, 24 h) and cultured under 20% or 1% oxygen conditions for 24 h. Cells were lysed and expression of HIF-1α, HIF-2α, Carbonic anhydrase IX (CAIX) and housekeeping gene B-actin assessed by western blotting, uncropped blots are available in Supplementary Figs. 3 and 4 (c) and mRNA levels of HIF-1α, HIF-2α and several HIF target genes (CAIX, BNIP3, VEGFA and GLUT1) quantified by qPCR (d). HepaRG cells encoding wild type HBx were incubated with Tet (50 µM, 24 h) and cultured at 20% or 1% oxygen for 24 h. The hypoxic cultures were returned to 20% oxygen. After 10 or 20 min, cells were lysed and screened for HIF-1α or HIF-2α and housekeeping gene β-actin expression by western blot, uncropped blots are available in Supplementary Fig. 5 (e). The data is shown from a single experiment and is representative of three independent experiments and represents mean ± standard deviation. Normality distribution was assessed by D'Agostino-Pearson test; 2-way-ANOVA with Bonferroni correction was applied with p < 0.05 deemed as significant.