Fig. 3

rt269L genotype C HBV infection led to enhanced autophagy induction. A, B Detection of autophagy induction by fluorescence microscopy. A HepG2 cells were transfected with the rt269L or rt269I vector. Cells were immunostained with antibodies against LC3B (green, Alexa 488) and HBV-pol (red, Alexa 594). The mean intensity of fluorescence (MIF) was analyzed. B Detection of LC3 puncta after transfection of the EGFP-LC3 vector. LC3 is recruited to autophagosomes, forming a punctate structure, as shown by green dots. The LC3-positive puncta in rt269L- or rt269I-transfected cells were analyzed in the absence or presence of bafilomycin A1 or a PERK inhibitor (GSK2656157, 10 µM). C EGFP-LC3 assay by flow cytometry. EGFP-LC3B-positive HepG2 cells cotransfected with mock, rt269L, or rt269I vectors were detected by flow cytometry. D Western blot analysis of the autophagy marker proteins LC3 and Beclin 1. The relative intensity of LC3II/LC3I staining was analyzed. **p < 0.01, ***p < 0.001. E Detection of autophagosomes and autolysosomes in HepG2-NTCP-C4 cells infected with rt269L or rt269I HBV virions, assessed by TEM. The arrow denotes autophagic vacuoles. Scale bar, 1 µm. F RT–qPCR analysis of Beclin1 and LC3 mRNA 10 h after HepG2-NTCP-C4 cell infection with PBS (Con), or rt269L or rt269I HBV virions. *p < 0.05, **p < 0.01, ***p < 0.001