Figure 3.

Overexpression of ATG5-ATG12 reduces FMDV yields and knock down of ATG5-ATG12 increases FMDV yields. (a-c) Overexpression of ATG5-ATG12 suppresses FMDV replication. PK-15 cells were transfected with vector or Flag-ATG5-ATG12 plasmid for 24 h, followed with FMDV infection (MOI=1) for 1, 3, 5, and 7 h. (a) Total RNAs were extracted and the levels of viral mRNA were determined with quantitative real-time RT-PCR analysis. (b) Protein samples were subjected to Western blot analysis to detect the expression of Flag-ATG5-ATG12 and viral proteins VP0, VP1, VP2, and VP3. (c) Supernatants were collected and subjected to TCID₅₀ assay to measure virus titer. (d-f) Knock down of ATG5-ATG12 enhances FMDV yields. PK-15 cells were transfected with control siRNA (NC) or siRNA targeting to ATG5 and ATG12 for 36 h, followed with FMDV infection (MOI=1) for 1, 3, 5, and 7 h. (d) Total RNAs were extracted and the levels of viral mRNA were determined with quantitative real-time RT-PCR analysis. (e) Protein samples were subjected to Western blot analysis to detect the expression of Flag-ATG5-ATG12 and viral proteins VP0, VP1, VP2, and VP3. (f) Supernatants were collected and subjected to TCID₅₀ assay to measure virus titer. (g) ATG5-ATG12 inhibits viral replication in a dose-dependent manner. PK-15 (1.2 × 10⁶ cells each well) cells were transfected with increasing dose of Flag-ATG5 and Flag-ATG12 plasmid (0, 0.25, 0.5, 1 or 2 μg), followed with FMDV infection (MOI=1) for 5 h. Protein samples were subjected to Western blot analysis to detect the expression of Flag-ATG5-ATG12 and viral proteins VP0, VP1, VP2, and VP3. Above data are representative of three independent experiments. Graphs show mean±S.D.; n=3. *P<0.05; **P<0.01