Fig. 5: Silencing Rubicon enhances autophagic flux to mitigate FFA-induced lipid metabolism dysfunction.

a Western blot assessment of autophagy-linked proteins, LC3 and p62, under diverse treatment regimes. b RT-PCR evaluation of gene expressions implicated in both autophagy and lipid metabolism in response to different treatments (n = 3). c IF visualization and d quantification of Lysotracker/Bodipy co-staining to detect lipophagy status by assessing co-localization of lipid droplets and lysosomes under varying conditions. White arrows highlight co-localization points of Lysotracker and Bodipy (n = 8). e IF detection and f quantification of LC3/Bodipy overlap to evaluate the co-localization of autophagosomes with lipid droplets, indicative of lipid degradation, under different treatments, with white arrows marking LC3-Bodipy co-localization (n = 8). g hepatocytes transfected with mCherry-GFP-LC3B virus and exposed to multiple treatments undergo IF analysis and h quantification to ascertain LC3B autophagic flux dynamics (n = 3). i IF and j quantification of LC3/LAMP1 co-staining to appraise autophagosome-lysosome co-localization, reflecting autophagic activity under varying conditions, where white arrows denote LC3-LAMP1 co-localization sites (n = 3). k TEM analysis provides insights into the state of autophagosomes and lipid droplets within cells, with red arrowheads pinpointing autophagosomes and yellow stars identifying lipid droplets. Statistical significance designated by * p < 0.05, ** p < 0.01, and *** p < 0.001, insignificance was not represented. Error bars indicate means ± SEM in (b, d, f, h, and j). Data were analyzed by oneway ANOVA followed by Tukey’s test. Source data are provided as a Source Data file.