Fig 2: Hypertrophic and inflammatory responses in TAC-operated Rubicon−/− hearts. (a) Representative hematoxylin–eosin-stained sections of Rubicon+/+ and Rubicon−/− hearts. Right panel, quantitative analysis of cross-sectional area of cardiomyocytes. Scale bar: 50 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upmu$$\end{document}μ m. N = 3. (b) Representative Azan-Mallory-stained sections of the hearts. Right panel, quantitative analysis of fibrotic area. Scale bar: 200 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upmu$$\end{document}μ m. N = 3. (c) Expression levels of mRNAs related to cardiac remodeling. Data were normalized to the Gapdh content and are expressed as fold increase over levels in the sham-operated Rubicon+/+ group. N = 5. (d) Representative confocal images of TUNEL-positive cardiomyocytes. Right panel, quantitative analysis of TUNEL-positive cardiomyocytes. N = 3. (e) Representative images of immunohistochemical analysis of CD45, CD68, Ly6G and CD3. Scale bar: 100 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upmu$$\end{document}μ m. Right graphs show quantitative analysis of each infiltrating inflammatory cell type. N = 3. (f) Expression levels of cytokine mRNAs. Data were normalized to the Gapdh content and are expressed as fold increase over levels in sham-operated Rubicon+/+ group. N = 5. Open and closed bars indicate Rubicon+/+ and Rubicon−/−, respectively. Data are expressed as the mean ± s.e.m. One-way ANOVA was used for analysis.

Fig 3: HCV infection increased Rubicon gene expression in the livers of human hepatocyte chimeric mice. Human hepatocyte chimeric mice were infected with HCV (genotype 1b) patient serum for 8 weeks. HCV-infected chimeric mice were treated and achieved sustained viral response (SVR). (A) The mRNA expression levels of Rubicon, Atg7 and type 1 interferon-related genes in the livers of control uninfected chimeric mice (n = 5), HCV-infected chimeric mice (n = 5) and SVR mice (n = 4). (B) Correlation diagram between the Rubicon gene and the ISG15, ISG56 and Mx1 genes. *p < 0.05.

Fig 4: HCV infection suppressed hepatocyte autophagy through upregulation of Rubicon expression. (A–E) Huh7.5.1 cells were infected with HCV (JFH-1) and then harvested at the indicated times. Western blot analysis of the HCV core, HCV NS5A and LC3 (A). Representative confocal microscopy images of Huh7.5.1 cells and JFH-1-infected Huh7.5.1 cells at 72 h after inoculation (B). Immunofluorescent staining was performed with DAPI (blue) and a monoclonal antibody against LC3 (green). LC3 turnover assay to determine the autophagic flux index using bafilomycin, a late-stage inhibitor of autophagy, at 72 h after inoculation (C); n = 3 per group. Western blot analysis of HCV core, HCV NS5A and autophagy-related proteins (D). The mRNA expression level of Rubicon (E). (F) Huh7.5.1 cells were transfected with siRNA against Rubicon (siRub) or negative control siRNA (siNC) for 72 h and infected with JFH-1 for 48 h. LC3 turnover assay to determine the autophagic flux index using bafilomycin, a late-stage inhibitor of autophagy (n = 3 per each). *p < 0.05.

Fig 5: GATA4 is selectively degraded in Sertoli cells in a lysosome-dependent manner.(A) Immunoblotting to detect GATA4 in Luciferase or Rubicon knockdown 15P-1 cells treated with or without 125 nM bafilomycin A1 for 4 h. Knockdown was performed for 48 h. n = 4. (B) Quantification of the relative GATA4 levels in (A). (C) Immunoblotting to detect the indicated proteins in 15P-1 cells treated with 125 nM bafilomycin A1 for the indicated times. n = 4. (D) Quantification of relative GATA4 levels in (C). (E) Quantification of relative WT1 levels in (C). (F) Immunoblotting to detect the indicated proteins in nuclear and cytoplasmic fractions of Luciferase or Rubicon knockdown 15P-1 cells. Knockdown was carried out for 48 h. n = 3. (G) Quantification of relative GATA4 levels in (F). (H) Immunoblotting to detect the indicated proteins in the nuclear and cytoplasmic fractions of 15P-1 cells. The cells were treated with 125 nM bafilomycin A1 for the indicated times. n = 3. (I) Quantification of relative GATA4 levels in (H). Error bars indicate means ± SEM. Data were analyzed by two-tailed Student’s t-test (G), one-way ANOVA followed by Tukey’s test (B, D, E, I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. N.S., not significant.