Fig. 5

Pan-caspase inhibitor inhibits nucleo-cytoplasmic translocation of IAV-nucleoprotein (NP). (A) Human airway epithelial cells were infected with 0.1 MOI IAV (H1N1) PR/8/34 strain and treated with vehicle control or 20 µM Q-VD-Oph. The inhibitors were added on apical and basal sides 3 h prior to viral infection. The inhibitors were removed from the apical side but were kept with media in the basal side for the whole experimental period. Protein lysates were analyzed for (A) caspase 3 and (B) PARP1 cleavages by Western blot. (C) Cells were subjected to immunostaining using a specific anti-viral NP antibody and secondary antibody conjugated to Dylight-649 24 h after infection. Cells were mounted with DAPI containing Fluormount-G for nuclear staining and analyzed with fluorescent microscopy. Percentage of IAV-infected cells with cytoplasmic localization were analyzed. Experiments were performed in triplicates, and the localization of viral NP in infected cells was quantified by counting at least 100 cells per experiment. n = 14/group, Error bars indicate mean ± SEM. Scale bar = 10 μm. (D) Protein lysates were analyzed for cleavage of HA and NP cleavage by Western blot. Experiments were performed in triplicates and the immunoblots are representative of at least three separate experiments. Q-VD-Oph, quinoline-Val-Asp-difluorophenoxymethylketone. p < 0.05 was considered significant