Fig 2: Role of lysosomal rupture/dysfunction in HIV- and ethanol-metabolism-induced apoptosis in hepatocytes: (1) Combined treatment with HIV and AGS triggers ROS and acetaldehyde release, which mediates lysosome leakage; (2) HIV/ethanol metabolism triggers the release of DRAM1, which also induces lysosome leakage; (3) Cathepsin B leaked out from damaged lysosome and diffused into the mitochondrion to initiate the intrinsic apoptotic pathway; (4) Caspases 9 and 3 become cleaved, leading to hepatocyte apoptosis; (5) Alcohol metabolites inhibit lysosome biogenesis factor TFEB, hence impairing the compensation of damaged lysosomes; and (6) Both lysosome damage and impaired lysosome biogenesis lead to HIV–ethanol-metabolism-induced lysosome dysfunction, which triggers apoptosis.

Fig 3: AGS and HIV enhance ZKSCAN3 but suppress TFEB target genes. (A,B) Cht-L, and T-L proteasome peptidase activities were detected in cell lysates by in vitro assay using fluorimetric substrates (C) Immunoblot analysis of ZKSCAN3 protein expression in RLW nuclear fraction. Equal (20 µg) amounts of protein were loaded in each lane. ß-actin was used as an internal control. (D) Quantification of immunoreactive bands of ZKSCAN3 from 3 independent experiments. (E) LAMP1 and cathepsin D mRNA expression detected by RT-PCR analysis. GAPDH mRNA was used as an internal control. Data are from 3 independent experiments, presented as the mean ± SEM. Bars marked with the same letter are not significantly different from each other; bars with different letters are significantly different (p = 0.05).

Fig 4: HG-induced autophagy blockage was due to TFEB-mediated lysosomal dysfunction in LECs. LECs were treated with various concentrations of glucose (5, 10, 25, and 50 mM) for 3 days. (A–C) Western blot analysis showed the effects of glucose on the changes of mesenchymal markers (a-SMA and FN) and autophagic markers (LC3-I/II, P62, p-mTOR, mTOR, ATG7, and ATG5). (D) Representative fluorescence images of mCherry-EGFP-LC3 puncta within LECs and quantification. Yellow puncta: autophagosomes, red-only puncta: autolysosomes. (E) Western blot analysis showed the expression of lysosome-associated proteins (ubiquitin, TFEB, ZKSCAN3, Pro-CTSB, and LAMP1) upon HG stimulation. (F) Immunofluorescence staining of LAMP1 puncta within LECs. (G) Lysotracker assay showed reduced lysotracker staining with increased concentrations of glucose. Relative protein levels of total proteins were standardized to the expression of ß-Actin. All the data were shown as mean ± standard deviation. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 5: DAF-16/FOXO and HLH-30/TFEB co-regulate a large number of genes, in particular genes that influence aging. a C. elegans with the genotypes wild type, daf-2(e1370ts), daf-16(mu86lf); daf-2(e1370ts), and daf-2(e1370ts); hlh-30(tm1978lf) were grown to the L4 stage at 15 °C, then shifted for 12 h to 25 °C, harvested, and their transcriptomes determined by mRNA-seq. The scatter plots on the left show the genes significantly upregulated and the scatter plots on the right the genes significantly downregulated in daf-2(e1370ts) compared to wild type animals. b C. elegans with the genotypes wild type, glp-1(e2141ts), daf-16(mu86lf); glp-1(e2141ts), and glp-1(e2141ts); hlh-30(tm1978lf) were grown from the L1 stage at 25 °C, harvested as young adults, and their transcriptomes determined by mRNA-seq. The scatter plots on the left show the genes significantly upregulated and the scatter plots on the right the genes significantly downregulated in glp-1(e2141ts) compared to wild type animals. c C. elegans with the genotypes wild type, daf-16(mu86lf), and hlh-30(tm1978lf) were grown to young adulthood at 20 °C, then harvested, and their transcriptomes determined by mRNA-seq. The Venn diagrams illustrate the number of genes significantly regulated in the mutants compared to wild type as well as their overlap. d Based on the data from a, these Venn diagrams illustrate the number of genes significantly regulated in the double-mutants compared to daf-2(e1370ts) as well as their overlap. e Based on the data from b, these Venn diagrams illustrate the number of genes significantly regulated in the double-mutants compared to glp-1(e2141ts) as well as their overlap. (Significance of gene expression changes in a–e was determined by Cuffdiff, using an FDR of 0.05. Significance of gene list overlaps in c–e was determined by Fisher’s exact test.) f–h GO-term enrichment analyses, conducted on the co-activated and co-repressed genes shown in c–e. Only GO-terms of significant enrichment are shown (DAVID score = 1, *: here the term aging was amongst the 12 most enriched GO-terms, but its score was only 0.62 and therefore below the significance threshold). Arrows highlight any aging-related GO-terms, in particular stress responses, protein homeostasis, and aging itself