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. 2021 Mar 12;12:1658. doi: 10.1038/s41467-021-21821-0

Fig. 4. REV-ERBα regulates HBV transcription.

Fig. 4

a dHepaRG cells were infected with HBV for 6 days followed by SR9009 treatment (10 or 20 µM) for 48 h. HBV parameters: cccDNA, pC/gRNA, HBeAg and HBsAg were determined by qRT-PCR or ELISA assays. In all cases, data are expressed relative to untreated (Ctrl) cells (mean ± SEM, n = 6, Kruskal–Wallis ANOVA with Dunn’s test). b HBV transcripts in the SR9009 treated dHepaRG cells were quantified by northern blotting. Densitometric analysis quantified pC/pgRNA and preS1/S2 transcripts and was normalized to the 28 S and 18 S rRNA loading controls. c HepG2-NTCP cells were transfected with HBV EnhI/II-BCP or Bmal1 promoter luciferase reporters for 48 h and treated with SR9009 for 24 h and promoter activity quantified by measuring luciferase activity (mean ± SEM, n = 4–6). d HepG2-pEpi cells were treated with SR9009 (20 µM) for 48 h. HBV parameters: pC/pgRNA, HBsAg, HBeAg and extracellular particle HBV DNA were determined by qRT-PCR or ELISA assays. The reverse transcriptase inhibitor Entecavir (ETV, 1 μM) was included as positive control for the genesis of de novo particles and is represented by the dashed line. In all cases, data are expressed relative to untreated (Ctrl) cells (mean ± SEM, n = 4, Mann–Whitney test, Two-sided). e Parental (WT) or Rev-erbα KO HepG2-NTCP cell lysates were assessed for REV-ERBα expression together with housekeeping gene β-actin by western blotting. Total RNA was extracted and Bmal1 mRNA levels measured by qRT-PCR. Data are expressed relative to WT cells (mean ± SEM, n = 5, Mann–Whitney test, Two-sided). f WT or Rev-erbα KO cells were co-transfected with HBV BCP luciferase reporter and Renilla luciferase reporter and 48 h post transfection the cells lysed and luciferase activity quantified. Data are expressed relative to WT (mean ± SEM, n = 5, Mann–Whitney test, Two-sided) and the data normalized for transfection efficiency. g WT or Rev-erbα KO HepG2-NTCP cells were infected with HBV and pC/pgRNA measured by qRT-PCR 72 h later. Data are expressed relative to WT (mean ± SEM, n = 6, Mann–Whitney test, Two-sided). *p < 0.05, **p < 0.01. Data are provided in the accompanying Source Data file.