Figure 2

NSP2-induced autophagy depends on aggresome formation. A The cytotoxicity of nocodazole was determined using the CCK-8 assay. Monolayers of Marc-145 cells and 293T cells in 96-well plates were treated with nocodazole at different concentrations for 48 h, after which the CCK-8 reagent was added to each well. After incubation for 2 h at 37 °C, cell viability was detected by measuring the absorbance at 450 nm. B 293T cells were transfected with empty vector and GFP-NSP2. After 6 h, cells were left untreated or treated with nocodazole (0.08, 0.16, or 0.32 µg/mL) for 4 h. At 24 h after transfection, cell lysates were used for western blot analyses with antibodies against β-actin, GFP, p62, and LC3. C 293T cells were transfected with empty vector and GFP-NSP2. After 6 h, cells were left untreated or treated with nocodazole (0.32 µg/mL) for 4 h. Cell lysates were harvested at 12 h, 24 h, and 36 h after transfection and analyzed by western blot using antibodies against β-actin, GFP, p62, and LC3. D Marc-145 cells were infected with HP-PRRSV (strain TA-12) at a multiplicity of infection of 0.1 for 1 h at 37 °C, after which the medium was replaced with maintenance medium containing 0.08, 0.16, or 0.32 μg/mL nocodazole. Cells were harvested 24 h later and analyzed by western blot with a monoclonal antibody (6D10) targeting the PRRSV N, or anti-β-actin, anti-GFP, anti-p62, and anti-LC3 antibodies.