Figure 2

ORFV-CL18 induces autophagic flux and the formation of autophagosomes and autolysosomes. A OFTu cells were mock infected, infected with ORFV-CL18, treated with 1 nM bafilomycin A1 (Baf A1) or treated with 1 nM Baf A1, and then infected with ORFV (MOI = 10). At 60 hpi, the expression levels of the autophagy marker proteins LC3B and P62 were measured by Western blotting. GAPDH was used as the loading control. B The ratios of LC3-II to LC3-I and P62 to GAPDH in different treatment groups. LC3-II and LC3-I protein levels were quantified by ImageJ software, and the ratio of LC3-II to LC3-I is expressed as the mean ± SD (n = 3) (*P < 0.05, *** P < 0.001; one-way ANOVA). The p62 protein expression level was normalized to that of GAPDH using ImageJ software, and the results are expressed as the mean ± SD (n = 3) (**P < 0.01; one-way ANOVA). C Autophagosome and autolysosome formation was detected by fluorescence assay. Briefly, OFTu cells transfected with the mRFP-GFP-LC3 expression vector were mock infected, infected with ORFV-CL18 and incubated for 36 h, 48 h and 60 h or treated with 30 μM rapamycin (Rapa) and incubated for 60 h. After treatment, the formation of autophagosomes (yellow puncta) and autolysosomes (red puncta) was analysed via confocal laser scanning microscopy. Scale bar, 10 μm. D, E The fluorescence signals of mRFP-LC3 (red) and GFP-LC3 (green) were measured by ImageJ/FIJI 36 hpi D and 60 hpi E, respectively. F OFTu cells were mock infected or infected with ORFV-CL18. At 36, 48 and 60 hpi, the morphology of subcellular organelles was observed by transmission electron microscopy. Asterisks indicate double-membrane vesicles, and red arrows indicate ORFV virions. (M mitochondria, ER endoplasmic reticulum, N nuclei, Aps autophagosome, Als autolysosome, Ly lysosome.) (Scale bars of a, c, e and g, 1 μm; scale bars of b, d, f and h, 500 nm).