FIG 3.

Inhibition of autophagy reduced NDV replication on DF-1 cells. (A) Western blotting of the autophagy inhibited with wortmannin. DF-1 cells were pretreated with wortmannin (100 nM) for 4 h followed by infection of NDV Herts/33 at an MOI of 1. DMSO was used as a control. After 1 h of virus absorption at 37°C, the cells were further cultured in fresh medium in the absence or presence of wortmannin. At 12 and 18 h after infection with NDV, the cells were subjected to Western blotting using anti-LC3B and -P antibodies. β-Actin was used as a protein loading control. (B) Determination of the NDV replication on wortmannin-treated DF-1 cells. The extracellular virus yields were determined at 12 and 18 hpi and expressed as TCID₅₀/ml. Data are presented as means from three independent experiments. Significance is analyzed with two-tailed Student's t test. *, P ≤ 0.05. (C) Western blotting of the autophagy and NDV P expression on DF-1 cells inhibited with wortmannin at the concentrations of 100, 300, and 500 nM. The samples were collected at 18 h after infection with NDV at an MOI of 1. (D) NDV titers on DF-1 cells treated with wortmannin at different concentrations. The extracellular virus yields were determined at 18 hpi and expressed as TCID₅₀/ml. Data are presented as means from three independent experiments. Significance is analyzed with two-tailed Student's t test. *, P ≤ 0.05; **, P ≤ 0.01. (E) Western blotting of the autophagy inhibited with CQ. DF-1 cells were pretreated with CQ (50 μM) for 4 h followed by infection with NDV Herts/33 at an MOI of 1. Double-distilled water was used as a control. After 1 h of virus absorption at 37°C, the cells were further cultured in fresh medium in the absence or presence of CQ. At 18 and 24 h after infection with NDV, the cells were subjected to Western blotting using anti-LC3B and -P antibodies. β-Actin was used as a protein loading control. (F) Determination of the NDV replication on CQ-treated DF-1 cells. The extracellular virus yields were determined at 18 and 24 hpi and expressed as TCID₅₀/ml. Data are presented as means from three independent experiments. Significance is analyzed with two-tailed Student's t test. *, P ≤ 0.05; **, P ≤ 0.01. (G) Western blotting of the autophagy and NDV P expression on DF-1 cells inhibited with CQ at the concentrations of 25 and 50 μM. The samples were collected at 24 h after infection with NDV at an MOI of 1. (H) NDV titers on DF-1 cells treated with CQ at different concentrations. The extracellular virus yields were determined at 24 hpi and expressed as TCID₅₀/ml. Data are presented as means from three independent experiments. Significance is analyzed with two-tailed Student's t test. *, P ≤ 0.05; **, P ≤ 0.01. (I) DF-1 cells were transfected with either specific siRNA targeting Beclin 1 or scrambled siRNA. At 48 h after transfection, cells were infected with NDV Herts/33 at an MOI of 1. Samples were collected at 18 h after infection with NDV and subjected to Western blotting of Beclin 1 and NDV P expression levels using anti-Beclin 1 and -P antibodies. β-Actin was used as a protein loading control. (J) The extracellular virus yields were determined at 18 h postinfection and expressed as TCID₅₀/ml. Data are presented as means from three independent experiments. Significance is analyzed with two-tailed Student's t test. *, P ≤ 0.05.