Figure 3

TMUV NS2B and NS4A induce autophagy. A HEK293T cells were transfected with pCAGGS empty vector and 10 protein plasmids for 24, 36, 48, and 60 h. Samples were harvested for Western blotting analysis and immunoblotted for the proteins LC3, HA, and β-actin. Bands labeled by “★” indicated the expressed specific viral proteins. B Normalized LC3-II/β-actin intensity band ratio from the data in A. (C) HEK293T cells were transfected with pCAGGS empty vector and 10 protein plasmids for 30 h. To inhibit the autophagic degradation of LC3, cells were treated with CQ (40 µM) for 24 h and then harvested. D Normalized LC3-II/β-actin intensity band ratio from the data in C. E BHK-21 cells were co-transfected with pEGFP-LC3 and ten TMUV protein plasmids. Cells co-transfected with pEGFP-LC3 and pCAGGS vector plasmids were used as the negative controls, and the cells transfected with pEGFP-LC3 and treated with Rapa (200 nM) were as the positive controls. Cells were fixed and imaged for GFP fluorescence after transfection for 36 h. Scale bar, 10 μm. F Number of the GFP-LC3 puncta per cell. The GFP-LC3 puncta were counted in 40 cells per group. One-way ANOVA was performed to determine statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).