Fig. 2: GAL9 leads to the autophagic degradation of HBc. | Nature Communications

Fig. 2: GAL9 leads to the autophagic degradation of HBc.

From: Galectin-9 restricts hepatitis B virus replication via p62/SQSTM1-mediated selective autophagy of viral core proteins

Fig. 2

a GAL9 decreases the level of HBc protein. Immunoblotting (upper) and RT-PCR assays (lower) of HepG2 cells expressing HA-HBc and GFP-GAL9. b Bafilomycin A1 inhibits GAL9-induced HBc degradation. HepG2 cells were transfected with vectors encoding HA-HBc and GFP-GAL9. Cells were treated with bafilomycin A1 (BafA1, 100 µM) or MG132 (10 µM) 16 h prior to harvesting. c ATG5 depletion attenuates GAL9-induced HBc degradation. HepG2 transduced with control- (Ctrl) or ATG5-targeting siRNA were transfected with vectors encoding HA-HBc and GFP-GAL9. d GAL9 and HBc accumulation in cytoplasmic bodies. Confocal microscopic imaging of HepG2 cells expressing HA-HBc and HaloTag empty vector (upper) or HaloTag-GAL9 (lower). Nuclei were stained with DAPI. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. 2a. e GAL9 and HBc accumulation in intracellular membranes. Confocal micrographs show HepG2 cells expressing HA-HBc and HaloTag-GAL9. Intracellular membranes were stained with CellMask reagent. Expanded views are also shown. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. 2b. f GAL9 and HBc co-localize with LC3. Confocal micrographs show HepG2 cells expressing HT-GAL9, HA-HBc, and GFP-LC3. Expanded views are also shown. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. 2c. g IFN-β increases colocalization of HBc and LC3 in a GAL9-dependent manner. Confocal micrographs show HepG2 cells expressing HA-HBc in the presence or absence of IFN-β. Scale bar, 10 µm. Another view of the cell image is shown in Supplementary Fig. 2f. The graph on the right shows the percentage of cells in which HBc localized to LC3-positive puncta (n = 50 cells examined over three experiments, mean ± SD). **P = 0.0027, ****P < 0.0001, two-tailed unpaired t-test. Scale bar, 10 µm. Bar charts in a–c indicate the ratio of HBc over Vinculin, as determined by densitometry, and are presented as a mean ± SD (n = 3 experiments). ****P < 0.0001 (a, b), **P = 0.0011 (c), two-tailed unpaired t-test. Immunoblots and micrographs are representative of experiments with similar results (n ≥ 2). Source data are provided as a Source Data file.

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