FIG 7.
Pepstatin A restored the HCV NS5A-induced CMA-mediated lysosomal degradation of FLAG-HNF-1α. Huh-7.5 cells were plated at 1.0 × 105 cells/24-well plate and cultured for 12 h. Cells were infected with HCV J6/JFH1 at an MOI of 2. At 4 h postinfection, the cells were transfected with either pCAG-FLAG-HNF-1α or pCAG-FLAG-HNF-1α Q130A. After transfection, 20 μM pepstatin A was administered to the cells as indicated. At 6 days postinfection, the cells were stained with anti-FLAG MAb followed by Alexa Fluor 405-conjugated goat anti-mouse IgG (blue), anti-NS5A PAb followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), and LysoTracker (red). These stained cells were examined using a Zeiss LSM 700 scanning laser confocal microscope and image software. Scale bars, 10 μm.