Fig. 2

Measurement of autophagic flux in BHK-21 cells infected with PFV. a BHK-21 cells were infected with PFV to analyze p62 protein expression by western blotting. After 1.5 h of virus absorption at 37 °C, the cells were further cultured in maintenance medium. The cells were infected with PFV at an MOI of 0.5. After PFV infection, cell samples were harvested at 0, 12, 24, 48, 72 and 96 hpi, and cell extracts were blotted with anti-p62 antibody. b BHK-21 cells were pretreated with the autophagy inhibitor CQ (50 μM) for 4 h, followed by infection with mock or PFV at an MOI of 0.5. After 1.5 h of virus absorption at 37 °C, the cells were further cultured in maintenance medium in the absence or presence of CQ. At 24 h of infection with mock or PFV, the cells were subjected to western blotting using anti-LC3 and anti-p62 antibodies. c BHK-21 cells transfected with ptfLC3 were infected with mock or PFV (MOI = 0.5). The cells were collected, fixed, and visualized at 24 hpi using a confocal microscope. Scale bars 100 μm. d Co-localization of the GFP-LC3 and LAMP1 proteins in BHK-21 cells infected with PFV and treated with CQ or DMSO. The cells were cotransfected with GFP-LAMP1 and RFP-LC3 for 12 h and pretreated with CQ (50 μM) or DMSO control. Then cells were infected with mock or PFV at an MOI of 0.5 for 24 h. The protein localization was observed using a confocal microscope. Scale bars 10 μm. Significance was analyzed with a two-tailed Student’s t test. ^ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001