Figure 5. AMPK is assuredly an upstream regulator of mTOR in BTV1-induced autophagy.

(a) Effects of Compound C treatment on LC3-II, CaMKKβ, phosphorylation and total levels of AMPK and mTOR. Cells were pretreated with Compound C (5 μM) or DMSO (Control) for 1 h, followed by BTV1 adsorption for 1 h. Then, the cells were cultured in the absence or presence of Compound C (5 μM). At 36 hpi, the protein levels were measured by western blotting. (b,c) BSR cells transfected with pEGFP-LC3 for 12 h were treated with 5 μM Compound C or BTV1, as before. The formation of GFP-LC3 punctae was analyzed, and the average number of green punctae per cell in each treatment was counted. (d) Titers of BTV1 produced by Compound C-treated BSR cells. Cells were pretreated and infected as described in (a). Extracellular and intracellular virus yields are shown as TCID₅₀/mL at 24 hpi and 36 hpi. **P < 0.01, significantly different. (e) Western blotting analysis of the effectiveness of AMPK knockdown in BSR cells. BSR cells were transfected with control siRNA (siNC) or AMPK-specific siRNA for 36 h and then analyzed by Western blotting. (f) Knockdown of AMPK affects the activity of mTOR and autophagy in BTV1-infected cells. BSR cells were transfected with either control siRNA or AMPK-specific siRNA, then infected with BTV1. At 36 hpi, cells were harvested, and western blotting was performed. (g) Extracellular and intracellular virus yields in BSR cells transfected with siRNA against AMPK. Data are shown as TCID₅₀/mL at 24 hpi and 36 hpi. *P < 0.05, **P < 0.01.