Figure 1.

FMDV infection induces ER stress and autophagy activity. (A) The Bip, LC3, P62, and FMDV structure proteins were detected by Western blot at indicated time points. The right graph shows the band intensities of specific proteins by densitometry using ImageJ software. The levels of related proteins were normalized to β-actin; data were expressed as means ± S.D of three experiments. Every treatment group compared with control groups, respectively; *p < 0.05, **p < 0.01. (B) FMDV-infected and TG-treated cells were fixed at 9 hpi, and mock-infected cells were used as a negative control. Cells were then imaged using confocal microscopy with a ×100 oil-immersion objective lens to detect the aggregation of calnexin. Calnexin was stained with TRITC, FMDV stained with FITC, and nuclei were stained with DAPI. Scale bars: 7.5 μm. (C) TG-treated and FMDV-infected PK-15 cells were transfected with GFP-LC3 recombinant plasmid and fixed at 9 hpi for immunofluorescence. The GFP-LC3 puncta aggregation was observed under a laser confocal microscope. FMDV and nuclei were stained with TRITC and DAPI, respectively. Scale bars: 7.5 μm. The puncta quantification was determined by the cells with LC3 fluorescent puncta normalized to the number of nuclei. (D) PK-15 cells were pretreated with different concentrations of ER stress inhibitor 4-PBA for 3 h and infected with FMDV for 5 h. Cells were collected for detecting the levels of Bip, LC3, and P62 by Western blot. The levels of related proteins were normalized to β-actin; data were expressed as means ± SD of three experiments. Every treatment group compared with control groups, respectively; *p < 0.05, **p < 0.01, ***p < 0.001. (E) the GFP-LC3 puncta aggregation was imaged by laser confocal microscope. FMDV was stained with TRITC and nuclei with DAPI. Scale bars: 7.5 μm. The puncta quantification was determined by the cells with LC3 fluorescent puncta normalized to the number of nuclei.