Fig. 2

Asiatic acid decreased HBx stability. (A) HepG2-NTCP or Huh-7 cells transfected with 3xFlag-HBx plasmids were exposed to 20 µM asiatic acid, and then subjected to 10 µg/ml cycloheximide at the indicated times. HBx was examined by western blotting analysis using anti-Flag antibody; (B) HepG2-NTCP cells transfected 3×Flag-HBx plasmid were exposed to 20 µM asiatic acid, and then subjected to ubiquitin-proteasome inhibitor MG132 (10 µM) or autophagy pathway inhibitor CQ (50 µM) treatment for 8 h before the cells were harvested. The levels of the proteins were examined by western blotting analysis, and β-actin was used as the loading control; (C) SQSTM1, LC3-I, LC3-II (autophagy-related markers) were detected by western blotting assay. (D) Huh-7 cells were transfected with GFP-LC3 and subjected to 20 µM asiatic acid, the expression level of HBx and LC3 puncta were visualized by immunofluorescent staining. Scale bar = 10 μm. Representative data are from at least three independent experiments. Data are shown as mean ± SD, *P < 0.05; **P < 0.01