Fig. 4

ER stress-related UPR signaling was activated as PFV infection progressed. a BHK-21 cells were infected with PFV to analyze ATF6, JNK and PERK phosphorylation and IRE1 activation by western blotting. Cells were infected with PFV at an MOI of 0.5. After PFV infection, cell samples were harvested at 0, 24 and 48 h, and cell extracts were evaluated by western blotting. β-actin was used as the loading control. b BHK-21 cells were seeded in 6-well plates and infected with PFV for 24 h (MOI = 0.5). Then, the total RNA (2 μg) was reverse transcribed to cDNA. Q-PCR was used to examine the relative expression (normalized to β-actin) of ER stress sensors such as CHOP, GADD34, ATF4, and GRP78. c BHK-21 cells were seeded in 6-well plates and infected with PFV for 0, 24 and 48 h (MOI = 0.5). The mRNA levels of spliced XBP1 and unspliced XBP1 were measured by RT-PCR. DNA agarose gel electrophoresis revealed the mRNA levels of spliced XBP1 and unspliced XBP1 in the presence or absence of PFV infection. d Western blotting was used to analyze the expression of CHOP and JNK signaling in PFV-infected cells. At 0, 12, 24, 34, 48 h after infection with PFV (MOI = 0.5), the cells were subjected to western blotting with the indicated antibodies. e Western blotting was used to analyze the expression of LC3 and JNK signaling in PFV-infected cells in the absence or presence of the ER-stress inhibitor 4-PBA. BHK-21 cells were pretreated with 4-PBA for 4 h, followed by infection with PFV (MOI = 0.5). Then cells were further cultured in maintenance medium in the absence or presence of 4-PBA. At 48 h after infection with PFV, the cells were subjected to western blotting with the indicated antibodies. As the control groups, BHK-21 cells were infected with mock or PFV (MOI = 0.5). Then cells were further cultured in maintenance medium for 48 h. f The graphical illustrated principle signaling pathways involved in PFV-induced autophagy via the ER stress-related UPR and the summary of regulation between PFV replication and PFV-induced autophagy. Significance was analyzed with a two-tailed Student’s t test. ^ns P > 0.05, **P < 0.01, ***P < 0.001