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Figure 3 | Veterinary Research

Figure 3

From: Orf virus induces complete autophagy to promote viral replication via inhibition of AKT/mTOR and activation of the ERK1/2/mTOR signalling pathway in OFTu cells

Figure 3

ORFV induces autophagy by inhibiting the mTOR signalling pathway. 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-P70S6K (T389/T412), P70S6K, P62, Rheb 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-P70S6K/P70S6K D, P62/GAPDH E, Rheb/GAPDH F and LC3-II/LC3-I G. The results 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 rapamycin (Rapa) or treated with 30 μM Rapa, infected with ORFV (MOI = 10) and cultured for 60 h. After treatment, the expression levels of p-mTOR (S2448), mTOR and LC3B were measured by Western blotting. GAPDH was used as the loading control. I LC3-II/LC3-I and p-mTOR/mTOR protein levels were quantified by ImageJ software, and the ratios are expressed as the mean ± SD (n = 3) (*P < 0.05, **** P < 0.0001; one-way ANOVA). J The recombinant vector pCMV-C-Flag-Rheb carrying the Rheb gene and its mutant pCMV-C-Flag-RhebQ64L were identified by Western blotting. GAPDH was used as the loading control. K OFTu cells were mock infected, infected with ORFV-CL18, transfected with pCMV-C-Flag or pCMV-C-Flag-RhebQ64L or transfected with pCMV-C-Flag-RhebQ64L, infected with ORFV (MOI = 10) and incubated for 60 h. After treatment, the expression levels of the autophagy marker proteins LC3B, Rheb, mTOR and p-mToR were measured by Western blotting. GAPDH was used as the loading control. L The ratios of LC3-II to LC3-I, Rheb to GAPDH and p-mToR to mTOR were measured, and the results are expressed as the mean ± SD (n = 3) (*P < 0.05, ** P < 0.01, **** P < 0.0001; one-way ANOVA).

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