Figure 5

TMUV NS2B and NS4A enhance complete autophagy. A HEK293T cells treated with CQ (40 µM) or not were transfected with pCAGGS empty vector, pCAGGS-NS2B-HA, or pCAGGS-NS4A-HA for 24, 36, 48, and 60 h. Samples were harvested for Western blotting analysis and immunoblotted for the proteins p62, LC3, and β-actin. B Normalized p62/β-actin and LC3-II/β-actin intensity band ratios from the data in A. C HEK293T cells treated with CQ (40 µM) or not were co-transfected with pCAGGS-Flag-p62 and pCAGGS empty vector, pCAGGS-NS2B-HA, or pCAGGS-NS4A-HA for 24, 36, 48, and 60 h. Samples were harvested for Western blotting analysis and immunoblotted for the proteins Flag, HA, and β-actin. D Normalized Flag-p62/β-actin intensity band ratio from the data in C. E BHK-21 cells were co-transfected with ptf-LC3 and pCAGGS empty vector, pCAGGS-NS2B-HA, or pCAGGS-NS4A-HA. Cells co-transfected with ptf-LC3 and pCAGGS vector plasmids were used as negative controls, and cells treated with Rapa (200 nM) were used as positive controls. Cells were fixed and imaged for GFP and RFP fluorescence after transfection for 48 h. Bars, 10 μm. F Number of LC3 punctate fluorescence per cell. The LC3 puncta were counted in 40 cells per group. Student’s t test and one-way ANOVA were performed to determine statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).