FIG 1.

HBsAg and HBxAg expressions induce the accumulation of autophagosomes in Huh7 cells. (A) Huh7 cells were transfected with pCIneo, pCIHBenv(+), or pCIHBenv(−) or treated for 24 h with rapamycin (Rap) (200 nM). Seventy-two hours after transfection, the levels of LC3II, p62, and β-actin were evaluated by Western blotting. (B) Huh7 cells were transfected with pCIneo, pCIHBx-Flag, or pCIHBs-Flag. After 72 h of transfection, the levels of LC3II, p62, and β-actin were evaluated by Western blotting. Moreover, the expressions of HBxAg and HBsAg were verified by Western blotting using an anti-Flag antibody. Cells transfected with pCIneo were treated or not with rapamycin. (C) Huh7 cells were cotransfected with pCIneo, pCIHBx-Flag, or pCIHBs-Flag and peGFP-LC3. As a positive control for autophagy induction, Huh7 cells cotransfected with pCIneo and peGFP-LC3 were treated with rapamycin (100 μM) for 3 h. At 72 h posttransfection, cells were fixed and stained with an anti-Flag antibody (red) or DAPI (blue). (D) The numbers of GFP-LC3 puncta per cell were calculated. (E and F) Huh7 cells were cotransfected with peGFP-LC3 and the plasmid pCIneo, pCIHBx-Flag, or pCIHBs-Flag, expressing the HBV proteins (S-HBsAg or HBxAg) through a cytomegalovirus (CMV) promoter (E), or the plasmid pCIneo, pT7HB2.7x(−) (L-M-S), or pT7HB2.7 (L-M-S-X), expressing the HBV envelope proteins and HBxAg from HBV endogenous promoters (F). The percentage of GFP-LC3-positive cells with punctate LC3 was calculated in transfected cells at 72 h posttransfection. Calculation was based on the count in 100 cells under each condition.