Fig 2: The study of the mechanism by which atRA decreases Rubicon protein expression in Hepa1c1c7 cells. A–C: Western blotting of Rubicon in Hepa1c1c7 or fatty Hepa1c1c7 cells treated by 100 nM atRA with or without (A) 1 μM MG132, (B) 100 nM Bafilomycin A1 (BAF), or (C) 10 μg/ml cycloheximide (CHX) for 24 h. β-actin was used as an internal control (n = 3–4). Values are mean ± SEM. ∗P < 0,05, ∗∗P < 0,01 (one-way ANOVA with a Student-Newman post hoc test). NS, not significant. D: schematic illustration of the induction of lipophagy via reducing Rubicon expression by atRA in hepatocytes.

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: Cellular uptake, in vivo biodistribution and bioactivity assessments of sgRubicon‐LNP. A) Determination of LNP transfection capacity by flow cytometry analysis of DiR‐LNP in HEK293T, Hepa1‐6, HepG2, and Raw264.7 cells. B) IVIS representative images and quantitative analysis of DiR signal in major organs after the injection of DiR‐labelled sgRubicon‐LNP (n = 3). C) Western blot analysis of Rubicon expression in the liver of C57BL/6 mice treated with sgRubicon‐LNP. β‐Tubulin was used as a loading control (n = 3). The data shown in (B) and (C) were the mean ± SEM. * p < 0.05, ** p < 0.01 determined by two‐tailed t‐test.

Fig 5: Neuro2A cells expressing human APP-EGFP lacking Rubicon release more APP species to the culture media. (a) Neuro2A cells expressing APP-GFP were transfected with siRNA against the autophagy proteins Rubicon (siRub), Atg5 (siAtg5), and Atg16L (siAtg16L), an exosomal Rab, Rab27a (siRab27a) or control scrambled (siCtrl). Protein extracts were analyzed by Western blot using an anti-APP/amyloid β antibody (6E10). (b) Densitometric quantification of APP protein levels from protein extracts for each knockdown cell line. β-Actin expression was monitored as a loading control. (c) Culture media were collected 24, 48, and 72 h after transfection and analyzed by dot blot using the anti-APP/amyloid β antibody (6E10) and quantified (d). FC, fold change. (e) PVDF membranes containing culture media from each siRNA cell line plus a control with protein extract in RIPA 1x were incubated with α-Tubulin. (f) Autophagy flux in cells siRub was performed using Bafilomycin A1 (25 nM) and Chloroquine (25 µM) (Baf + CQ) as lysosome inhibitors for 2, 4, and 6 h before preparing protein extracts. Data are shown as means ± SEM. * p < 0.05; ** p < 0.01 as determined by repeated-measures ANOVA, n.s., no significance.