Gossypol Affects Viral Replication by Inhibiting Pseudorabies Virus Adsorption

Figure 5

Anti-PRV effect of gossypol was independent of the autophagy it induced. (a, b) NIH-3T3 (a) and PK-15 (b) cells were transfected with the GFP-RFP-LC3 plasmid and then treated with DMSO, gossypol (3 µM), or Rapa (1 µM) for 24 hr. The GFP and RFP fluorescence levels were detected under a confocal microscope. Scale bar, 10 µm. (c, d) NIH-3T3 (c) and PK-15 (d) cells were treated with gossypol (0, 0.1, 0.3, 1, or 3 µM) or Rapa (10 µM) for 24 hr, and then the expression of LC3-I, LC3-II, and P62 proteins was detected by western blot. (e) Mitophagy was detected after NIH-3T3 cells were treated with gossypol. NIH-3T3 cells were infected with pLVX-IRES-Puro-RFP-GFP-fis1 lentivirus, which has a mitophagy-indicating function, and 12 hr later, the cells were treated with complete medium containing the indicated concentration of gossypol (0, 0.1, 0.3, 1, or 3 µM) for 36 hr. The cells were fixed and sealed, and the occurrence of intracellular mitophagy was observed and photographed under a confocal microscope. Scale bar, 10 µm. (f) (Top) Detection of Atg5 in PK-15 Atg5^WT and PK-15 Atg5^KO cells by western blot; (Bottom) Atg5^WT and Atg5^KO PK-15 cells were treated with the indicated concentration of gossypol (0, 0.1, 0.3, 1, or 3 µM) and then infected with rPRV HN1201-EGFP-Luc (MOI = 0.01) for 24 hr. The proportion of EGFP-positive cells was determined by flow cytometry, and the results were normalized to those of the control group. (g) PK-15 cells were treated with the indicated concentration of gossypol (0, 0.1, 0.3, 1, or 3 µM) alone or cotreated with the indicated concentration of gossypol (0, 0.1, 0.3, 1, or 3 µM) and 3-MA (5 mM) and then infected with rPRV HN1201-EGFP-Luc (MOI = 0.01) for 36 hr. The proportion of EGFP-positive cells was determined by flow cytometry , , and .

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