Fig. 6.

PEDV ORF3 protein induces the autophagy that is dependent on ER stress response. (A) Vero cells were transfected with pEGFP-LC3, and then either transfected with pCMV-Myc-ORF3 or treated with rapamycin used for positive control, as well as pCMV-Myc-transfected cells used for negative control. At 12 h post transfection, cells were fixed, permeabilized and then probed with mouse monoclonal anti-Myc antibody, followed by TRITC-labeled goat-anti-mouse antibody (red). Nuclei were stained with DAPI (blue), and the GFP-LC3 punctate structures were visualized under confocal microscope. (B) Average numbers of LC3-positive vesicles per cell were quantified from three biological replicates. * P <  0.05; ** P <  0.01; *** P <  0.001. (C) Vero cells were transfected with pEGFP-LC3, and then either transfected with pEGFP-N1 or pEGFP-N1-ORF3 for 8, 16 and 24 h, and cell lysates were harvested and subjected to Western Blot probed with the mouse monoclonal anti-LC3 and anti-GFP antibodies, followed by the goat-anti-mouse-HRP antibody. Cellular β-actin was detected as the control protein. (D) Densitometry scans of the LC3-II brand of Western blot from (C). * P <  0.05; ** P <  0.01; *** P <  0.001. (E) The 293T cells were either treated with 4-PBA or not for 4 h, then transfected with pEGFP-N1-ORF3 or pEGFP-N1 for 24 h. Western Blot was performed for testing the endogenous LC3-I/LC3-II conversion with the mouse monoclonal anti-LC3 antibody followed by the goat-anti-mouse-HRP antibody. Cellular β-actin was employed as an internal loading control protein. (F) Densitometry scans of the LC3-II brand of Western blot from (E). * P <  0.05; ** P <  0.01; *** P <  0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).