However, it is not clear whether RIG-I activation by the recognition of a pathogen-associated molecular pattern regulates the autophagic process directly. The results of the present study provide evidence that RIG-I triggers autophagic flux upon recognition of its ligands. polyubiquitination of Beclin-1, which has been implicated in triggering autophagy. As deficient autophagy increases the type-I interferon response, the induction of autophagy by the RIG-I pathway might also contribute to preventing an excessive interferon response as a negative-feedback mechanism. test using GraphPad Prism 5 software. A value of * 0.05, ** 0.005, *** 0.0005 was considered significant. Results Activation of the RIG-I signaling pathway activates autophagy To investigate MK-1064 whether the recognition of viral RNA by RIG-I can trigger autophagy, HEK293T cells were transfected with the RIG-I agonist, polyI:C, a synthetic double-stranded RNA analog, or infected with Sendai virus (SeV). Both polyI:C transfection and SeV infection increased the level of LC3-II, a lipidated form of LC3 (Figure ?(Figure1A).1A). In both the experimental settings, the change was evident 2 h after stimulation (Figure ?(Figure1A).1A). Increased formation of autophagic vesicles was also observed in the polyI:C-transfected and SeV-infected cells compared with that in mock-infected cells (Figure ?(Figure1B),1B), as determined by Cyto-ID that can stain both autophagosomes and autolysosomes specifically (27). The increased autophagosome formation by polyI:C transfection or SeV infection was further confirmed by transmission electron microscopy. As shown in Figure ?Figure1C,1C, the number of dense black double-membrane structure vesicles increased in the polyI:C transfected or SeV infected cells. The formation of LC3 puncta by polyI:C or SeV infection also contributed to the modulation of autophagy by RIG-I signaling (Figure ?(Figure1D).1D). Not only the increase of autophagy flux but MK-1064 also the blockade of autophagosomal maturation can be SOCS-2 resulted in accumulation of LC3-II. Thus, LC3-II formation upon RIG-I activation was examined with or without bafilomycinA1, an inhibitor of the late phase of autophagy. BafilomycinA1 treatment resulted in further increase of LC3-II formation in polyI:C transfected or SeV infected cells compared to mock-treated samples. LC3-II formation was increased by PolyI:C transfection or SeV infection in a time-dependent manner MK-1064 regardless of bafilomycinA1 treatment (Figure ?(Figure1E).1E). Furthermore, the level of p62 decreased in a time-dependent manner in the polyI:C-transfected or SeV-infected cells, whereas the level was not noticeably changed in untreated samples (Figure ?(Figure1F).1F). These results indicated that the increased level of LC3-II was due to increased autophagy and not due to decreased phagolysosome formation. Open in a separate window Figure 1 RIG-I activation invokes autophagy. (A) HEK293T cells were transfected with polyI:C (2 g) or infected with Sendai virus (SeV) (200 HA U/mL) for the indicated hours. The cell lysates were analyzed by immunoblotting using antibodies for LC3 (LC3-I and LC3-II), phospho-IRF3 (p-IRF3), and -actin. (B) HEK293T cells were transfected with polyI:C or infected with MK-1064 SeV as in (A) and incubated for 12 h. Autophagosomes were labeled with cytoID-green reagent and observed by fluorescence microscopy. The bottom panels show staining with Hoechst dye to visualize nuclei of the cells. (C) HEK293T cells were transfected with polyI:C (10 g) or infected with SeV (200 HA U/mL) for 12 h. The cells were harvested, fixed, and subjected to transmission electron microscopy to observe autophagic vesicles (red arrows in the lower panels). The bottom panels show enlarged view of the boxed regions in the top panels. The bottom graph show that means of autophagic vacuoles in a cell determined from 5 different images. The data are presented as mean SE. (D) HEK293T cells were mock-treated, transfected with polyI:C or infected with SeV as in (A). After washing and fixation, LC3 puncta were visualized by staining with anti-LC3 antibody and FITC-labeled secondary antibody and observed by fluorescence microscopy. The number of puncta was counted and analyzed using the image J software. The bottom panel shows the mean number of LC3 puncta in a cell. The data are presented as mean SE. (E) HEK293T cells were transfected with polyI:C or infected with SeV and treated with or without bafilomycin (50 nM) for the indicated hours..