Fig. 3.

Asiatic acid affected cccDNA epigenetic modifications. (A-C) HepG2-NTCP cells were incubated with asiatic acid (20µM), following with or without CQ (50µM). The levels of total HBV RNAs and HBV 3.5-kb RNA were detected by real-time PCR using specific primers. The β-actin mRNA level was used as an internal control. The ratios of total HBV RNAs/cccDNA and HBV 3.5-kb RNA/cccDNA were calculated as indicators of cccDNA transcriptional activity; (D) The level of HBx associated with cccDNA in HBV-infected HepG2‐NTCP cells was examined by ChIP-qPCR assay. Cross-linked chromatin was immunoprecipitated with specific anti-HBx or anti-IgG antibody, followed by PCR quantification of HBV cccDNA using specific primers, with the host genes GAPDH and MYH6 used as controls; (E-G) The levels of H3K9me3, H3K27me3 and H3K4me3 associated with cccDNA, GAPDH or MYH6 promoter were analysed by ChlP assay with anti-H3K9me3, anti-H3K27me3, anti-H3K4me3 and anti-IgG, respectively. Representative data are from at least three independent experiments. Data are shown as mean ± SD, *P < 0.05; **P < 0.01