FIG 6.

PML is involved in the FoxO4-mediated epigenetic suppression of cccDNA. (A to C) Huh7 cells were transfected with control or PML siRNA. After 48 h, the cells were further transfected with FoxO4 and prcccDNA/Cre for another 48 h. (A) The protein level of PML was determined by Western blotting. (B) The recruitment of AcH3, H3K4me3, H3K9me3, and H3K27me3 onto rcccDNA was examined by ChIP assays, and data are shown as fold change versus empty vector-transfected cells after being normalized to input and control IgG. (C) The levels of HBV proteins (HBsAg and HBeAg), preC-pgRNA, and HBV DNA were determined by ELISA, qRT-PCR, and qPCR, respectively. (D to F) HepG2-NTCP cells were electrotransfected with control or PML siRNA. After 48 h, the cells were further transfected with FoxO4-Flag, followed by infection with HBV at 10³ vge/cell. At day 3 postinfection, the efficacy of PML knockdown was determined by Western blotting (D), and the recruitment of H3K4me3 and H3K9me3 onto cccDNA was determined by ChIP assays as in panel B (E). (F) At day 9 postinfection, the levels of HBsAg, preC-pgRNA, and HBV-DNA were determined by ELISA, qRT-PCR, and qPCR, respectively. The data are shown as means ± the SD of triplicates and are representative of three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001).