Fig. 6. The HA-containing structures can be classified as amphisomes.

a Production of LC3-II and the effects of protease inhibitors. MDCK cells were infected (IAV) or not infected (−) with IAV strain PR8 at 10 MOI for 1 h. After washing, the cells were cultured in the presence or absence of 20 μm PVF-tet for 2 h. The cells were treated (+) or not treated (−) with lysosomal protease inhibitor cocktail containing 30 μm E-64, 15 μm pepstatin A, and 20 μm leupeptin and further cultured for 13 h. The cell lysates were analyzed by western blot. b The effect of PVF-tet on the intracellular localization of LC3. MDCK cells were not infected or infected with IAV strain PR8 at 10 MOI for 1 h. After washing, the cells were cultured in the presence or absence of PVF-tet (20 μm) for 15 h. Localization of HA and LC3 was analyzed by immunocytochemical staining. c−d The effects of ULK1 or PIK3C3 knockout on the formation of the PVF-tet-induced HA-containing structure and the anti-IAV activity of PVF-tet. The expression levels of ULK1 in MDCK-derived ULK1-knockout clones (c, left upper panel) and PIK3C3 in MDCK-derived PIK3C3-knockout clones (left lower panel) were analyzed by western blot using specific antibodies. Parental MDCK cells or each MDCK-derived knockout clone was infected with IAV strain PR8 at 10 MOI for 1 h. After washing, the cells were cultured in the presence of 20 μm PVF-tet for 15 h and fluorescent images were analyzed using laser scanning confocal microscopy (c, right panel), or cultured for 23 h (MDCK cells) or 47 h (knockout clones) and cell viability was measured by a cytopathicity assay d. Data are presented as a percentage of the control value without infection (mean ± SEM of three independent experiments). **P < 0.01; ***P < 0.001 (compared with untreated cells by ANOVA followed by one-sided Dunnett’s test). n.s., not significant. Scale bars represent 20 μm. Source data including uncropped western blot images are provided as a Source Data file.