Figure 4

ORFV induces autophagy by inhibiting PI3K/AKT/mTOR signalling. A OFTu cells were mock infected or infected with ORFV-CL18. At 36, 48 and 60 hpi, the expression levels of p-mTOR (S2448), mTOR, p-TSC2 (S1387), TSC2, p-PI3K (Y467), PI3K, p-AKT (S473 and T308), AKT and LC3B were measured by Western blotting. GAPDH was used as the loading control. B–G The ratios of p-mTOR/mTOR B, p-TSC2/TSC2 C, p-PI3K/PI3K D, p-AKT (S473)/AKT E, p-AKT (T308)/AKT F, and LC3-II/LC3-I G. These protein bands were quantified by measuring the signal intensity with ImageJ, and the ratios are expressed as the mean ± SD (n = 3) (*P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; unpaired t test). H OFTu cells were mock infected, infected with ORFV-CL18, treated with 30 μM SC79 or treated with 30 μM SC79, infected with ORFV (MOI = 10) and incubated for 60 h. After treatment, the expression levels of p-mTOR (S2448), mTOR, p-AKT (S473), AKT and LC3B were measured by Western blotting. GAPDH was used as the loading control. I The ratios of p-mTOR/mTOR, p-AKT (S473)/AKT and LC3-II/LC3-I, which are expressed as the mean ± SD (n = 3) (**P < 0.01, *** P < 0.001, **** P < 0.0001; one-way ANOVA). J The recombinant vector pCMV-Myc-N-Akt was identified by Western blotting using an anti-Myc antibody. (K) OFTu cells were mock infected, infected with ORFV-CL18, transfected with pCMV-Myc-N or pCMV-Myc-N-Akt or transfected with pCMV-Myc-N-Akt, infected with ORFV (MOI = 10) and incubated for 60 h. After treatment, the expression levels of the autophagy marker proteins LC3B and Akt were measured by Western blotting. GAPDH was used as the loading control. L The ratios of LC3-II to LC3-I and Akt to GAPDH, which are expressed as the mean ± SD (n = 3). (*P < 0.05, *** P < 0.001, **** P < 0.0001; one-way ANOVA).