Fig. 2: B5G1 induces mitophagy in HepG2/ADM cells.

a After B5G1 treatment (6 μM) for 24 h, HepG2/ADM cells were stained with MDC (50 μM) or immunostained with a LC3 antibody. For the GFP-LC3 assay, HepG2/ADM cells were transfected with the GFP-LC3 plasmid for 24 h, followed by treatment with B5G1 (6 μM) for 24 h. The fluorescence was observed by a fluorescence microscope. Magnification: ×630, scale bar: 10 μm. b HepG2/ADM cells were treated with B5G1 as indicated, and LC3 expression level was then detected by western blotting. β-actin was used as a loading control. c After treatment with B5G1 (6 μM) for 24 h, the ultrastructure of HepG2/ADM cells was observed by a transmission electron microscopy. Magnification: ×8900; Scale bar: 500 nm. d HepG2/ADM cells were treated with B5G1 (6 μM) for 0, 12, 24, or 48 h and then stained with MitoTracker Red (200 nM) or MitoTracker Green (200 nM). The fluorescence was observed by a fluorescence microscope. Magnification: ×630; scale bar: 10 μm. e HepG2/ADM cells were exposed to B5G1 (6 μM) for the indicated times. Mitochondrial proteins expression levels were measured by western blotting. β-actin was used as a loading control. f HepG2/ADM cells were transfected with the mKeima-Red-Mito-7 plasmid, followed by treatment with B5G1 (6 μM) for 24 h. The Fluorescence was detected by a fluorescence microscope. Magnification: ×630; scale bar: 10 μm. g, h After treatment with B5G1 (6 μM) for the indicated times, HepG2/ADM cells were stained with MitoTracker red (200 nM) and immunostained with a LC3 or LAMP1 antibody. Mitochondrial colocalization with LC3 or LAMP1 was observed by a fluorescence microscope. Magnification: ×630; scale bar: 10 μm