Supplementary Material for: The Preventive Effect of the Traditional Japanese Herbal Medicine, Hochuekkito, against Influenza A Virus via Autophagy in vitro

<p><b><i>Background:</i></b> Hochuekkito (HKT), a traditional Japanese herbal medicine, enhances the immunity of elderly or weak individuals. It is also known to have preventive effects against influenza clinically. However, the detailed mechanisms of the preventive effects have not been clarified. We examined the relationship between the preventive effects of HKT and autophagy, a known stress response and quality control mechanism, using Madin-Darby canine kidney cells and influenza A/PR/8/34 (H1N1) virus. <b><i>Methods:</i></b> The effect of HKT on autophagy in influenza A virus (IAV)-infected cells was assessed by Western blotting and fluorescence microscopy using an RFP-GFP-LC3B sensor kit. <b><i>Results:</i></b> In Western blotting, treatment with HKT before IAV infection (pre-HKT) tended to induce autophagy in IAV-infected cells at an early stage of infection, eventually suppressing IAV-induced autophagy. Moreover, several autolysosomes, indicative of normal autophagosome-lysosome fusion, were observed in Pre-HKT cells transduced with RFP-GFP-LC3B but not in untreated IAV-infected cells. <b><i>Conclusions:</i></b> These findings indicated that IAV-mediated inhibition of the fusion of autophagosomes with lysosomes was prevented by HKT treatment before infection. According to these results, we propose that this phenomenon is one of the preventive effects of HKT against IAV.</p>