Fig. 2

The formation of autophagosomes induced by PRRSV NSP3 and NSP5. a Marc-145 cells were transfected with GFP-LC3 and plasmids encoding the individual PRRSV Nsps; each Nsp was cloned into an mCherry-N plasmid, and cells transfected with mCherry-N were used as a negative control. Twenty-four hours after transfection, cells were harvested and visualized under a fluorescence microscope. Nuclei were stained with DAPI, and the number of GFP-LC3 puncta, which is related to autophagy, was analyzed using GraphPad software. Scale bars: 10 μm. b Statistical analysis of the number of GFP-LC3 puncta in cells expressing mCherry-N and each Nsp; the numbers represent GFP-LC3 puncta per cell; data are presented as means ± SD, n = 30. c Cells were transfected with mCherry Nsp3-mCherry and Nsp5-mCherry for 24 h; the endogenous p62 protein was stained with a green fluorophore. Cell lysates were subjected to immunoblotting. Scale bars: 10 μm. d LC3 protein conversion. Marc-145 cells were mock infected; treated with HBSS for 4 h or transfected with mCherry, Nsp3-mCherry, or Nsp5-mCherry; and then harvested and lysed. Extracts were analyzed by western blotting using an anti-LC3 antibody. The ratio of LC3II/LC3I reflects the level of autophagy