Figure 4

EGCG, but not HBV, enhances autophagic degradation, which appears to be unfavorable for HBV replication. (a) The effect of HBV DNA transfection or starvation on p62 degradation in HepG2 cells. Cells were transfected with pUC19 or increasing dose of pHBV1.3 for 48 h. To determine the effect of starvation on p62 degradation, pUC19-transfected HepG2 cells were cultured in Earle's Balanced Salt Solution (EBSS) medium for 3 h. Cells were then subjected to western blotting using anti-p62. The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (b) The effect of EGCG on p62 protein level in HepG2 cells. Cells were treated with 25 or 50 μM of EGCG for 24 h and were then subjected to western blotting analysis as in panel (a). (c) The effect of EGCG on p62 mRNA level in HepG2 cells. Cells were treated as in panel (b), and p62 mRNA expression level was determined by quantitative reverse transcriptase-PCR, bars represent means±S.E.M (n=3). (d) The effect of HBV DNA transfection or starvation on GFP-LC3 processing. HepG2 cells stably expressing GFP-LC3 were transfected with pUC19 or increasing dose of pHBV1.3 for 48 h. To determine the effect of starvation on GFP-LC3 processing, pUC19-transfected cells were cultured in EBSS medium for 3 h. Cells were then subjected to western blotting with anti-GFP. The expression of GAPDH was used as a loading control. (e) The effect of EGCG on GFP-LC3 processing. GFP-LC3 stably transfected HepG2 were treated with 25 or 50 μM of EGCG for 24 h, and cells were then subjected to western blotting as in panel (d). (f) The effect of starvation on autophagosome formation and p62 degradation. HepG2.2.15 cells were cultured in EBSS medium for 3 h and were then subjected to western blotting using antibodies against LC3 and p62. The expression of GAPDH was used as a loading control. (g) The effect of starvation on HBV replication. HepG2.2.15 cells. were treated as in panel (f) and subjected to HBV DNA quantification using real-time PCR, bars represent means±S.E.M (n=3); *P<0.05. (h) The effect of ATG5 or ATG7 siRNA on EGCG-induced autophagy. HepG2.2.15 cells were electro-transfected with ATG5- or ATG7-specific siRNA. Seventy-two hours later, cells were subjected to the EGCG treatment for another 24 h. The expression of ATG5, ATG7, LC3, or p62 was determined by western blotting. GAPDH was used as a loading control. (i) The effect of ATG5 or ATG7 siRNA on EGCG-mediated inhibition of HBV replication. HepG2.2.15 cells were treated as in panel (h) and subjected to HBV DNA quantification using real-time PCR, bars represent means±S.E.M (n=6). *P<0.05