Fig. 4: HBsAg blocks the autophagosome–lysosome fusion in HepG2 cells.

A mCherry-GFP-LC3 expressing HepG2 cells were transfected with pHBV1.3, pHBV1.3-ΔHBs, or treated with CQ (10 μM). Cells were imaged by confocal microscopy. Scale bar: 10 μm. B Quantifying the percentage of yellow puncta/cell positive for both mCherry and GFP in cells. C HepG2 cells were transfected with pHBV1.3, pHBV1.3-ΔHBs plasmids or treated with CQ (10 μM). Cells were stained with AO and DAPI for 15 minutes and then detected by confocal microscopy. Scale bar: 20 μm. D Statistical analysis of relative AO-red intensity. E HepG2 cells were transfected with pHBV1.3, pHBV1.3-ΔHBs plasmids, or treated with BafA1 (100 nM). The co-localization of LC3 and LAMP1 was imaged. Scale bar: 10 μm. F Statistical analysis of co-localization between LC3 and LAMP1. The co-localization coefficient was represented as a percentage of puncta signals of LC3 that were positive for LAMP1. G HepG2 cells were transfected with pHBV1.3 or pHBV1.3-ΔHBs plasmids or stimulated with CQ or BafA1, and LC3B protein levels were analyzed by immunoblot. All experiments were repeated at least three times with consistent results. Bar graphs show the means ± SD (n = 3 biological replicates). ^**P < 0.01; ^***P < 0.001; ns not significant; using Student’s t-test.