ASFV K205R activates autophagy. (A) HeLa cells were transfected with empty vector or K205R-GFP plasmid for 24 h. LC3 was monitored with immunofluorescence analysis. Scale bar: 10 μm. (B) Quantification of LC3 puncta per cell from A (n = 30). *** p < 0.001. (C) 3D4/21 cells were transfected with K205R-HA plasmid as indicated for 24 h. LC3-I, LC3-Ⅱ, ATG5, ATG12, P62, Beclin-1, K205R-HA, and β-actin were assessed with immunoblotting analysis. (D) 3D4/21 cells were transfected with K205R-HA plasmid and treated with bafilomycin A1 (10 μM) as indicated for 24 h. LC3-I, LC3-Ⅱ, ATG5, ATG12, P62, Beclin-1, K205R-HA, and β-actin were assessed with immunoblotting analysis. (E) PK15 WT, ATG5−/−, and Beclin-1−/− cells were transfected with K205R-HA plasmid as indicated for 24 h. LC3-I, LC3-Ⅱ, ATG5, ATG12, P62, Beclin-1, K205R-HA, and β-actin were assessed with immunoblotting analysis. (F) 3D4/21 cells were transfected with K205R-HA plasmid and treated with GSK (10 μM) as indicated for 24 h. LC3-I, LC3-Ⅱ, ATG5, ATG12, Beclin-1, K205R-HA, and β-actin were assessed with immunoblotting analysis. (G) 3D4/21 cells were transfected with K205R-HA plasmid as indicated for 24 h. AKT, p-AKT, mTOR, p-mTOR, ULK1, p-ULK1 ser555, p-ULK1 ser757, K205R-HA, and β-actin were assessed with immunoblotting analysis.