Fig 2: Lysosomal protease leakage triggers mitochondrial protein degradation in macrophages.a Schematic showing the conditions tested in this study. b Immunoblots for MFN2, TOM20, TIM23, HSP60 and Citrate synthase (CS) in iPSDM stimulated with 0.5 mM LLOMe for 1 h, 100 µg/mL silica crystals or beads for 3 h or infected with Mtb WT or Mtb ?RD1 for 48 h and incubated with the indicated protease or proteasome inhibitors. Beta-actin (ACTB) levels were used as loading controls (repeated three times with similar results). c Immunoblots for mitochondrial proteins in iPSDM WT, ATG7 KO, PRKN KO and PRKN/ATG7 DKO stimulated with 0.5 mM LLOMe for 1 h and incubated in the presence or absence of the indicated inhibitors (repeated three times with similar results). d Immunoblots for mitochondrial proteins in BMM WT, CtsB KO, CtsL KO and CtsS KO stimulated with 0.5 mM LLOMe for 1 h. (repeated three times with similar results). e Representative images of iPSDM expressing the mitophagy reporter NIPSNAP and stimulated with 0.5 mM LLOMe for 1 h, 100 µg/mL silica crystals for 3 h, infected with Mtb WT for 48 h or treated with 20 µM CCCP for 3 h. f NIPSNAP mCherry only puncta evaluated by confocal microscopy, n = 30 cells examined per condition over three independent experiments. g TOM20+/PDHA1- and PDH+/TOM20- MDVs were quantified after the indicated conditions. Glucose oxidase (GO) was used at 50 mU/ml for 1 h as a positive control, n = 20 cells examined per condition over three independent experiments. h One-way ANOVA and Tukey post-test was used for multiple comparisons ***p = 0.001, ns no significant. Scale bars, 10 µm. Floating bar plots show minimum and maximum values, line at mean. Unprocessed blots and Source data are provided as a Source Data file.

Fig 3: GCN5L1 LKO impaired gluconeogenesis. a ​Glucose production was measured in the culture medium of primary hepatocytes isolated from GCN5L1 LKO and littermate controls at 5 h after treatment with or without 100 nM glucagon. ​Glucose production was normalized to protein content (n = 3 independent experiments). b HeLa cells were transiently transfected with Mt-GCN5L1 plasmid, followed by immunofluorescence staining and confocal analysis. Tom20 serves as mitochondrial marker. Scale bar, 5 μm. c Primary hepatocytes were isolated and transfected with adenoviral expression of vector control, GCN5L1, or Mt-GCN5L1. RNA was analyzed at 36 h after transfection (n = 3 independent experiments). Values are expressed as mean ± s.e.m. **P < 0.01 versus respective groups by Student’s t-test

Fig 4: Mitophagy is induced in CLP-induced AKI in mice. C57BL/6 mice (male, 8–10 weeks old) were subjected to CLP or sham-surgery for analysis 24 h later. Blood samples were collected for measurements of serum creatinine (A) and BUN (B). (C) Representative images of HE staining. (D) Percentage of damaged tubules. (E) Representative images of TUNEL staining, (F) Quantification of TUNEL positive cells. (G) Immunoblot analysis of TOM20, TIM23 and LC3 II/I. (H) Immunoblot analysis of PINK1 and PARK2. Mean ± SD. n ≥ 4. *P < 0.001 vs. the control group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig 5: Correlation between mtTFA protein level and mtDNA release from altered mitochondria in RNASEH2B- and RNASEH2A-mutated LCLs. (A) Representative Western blot and histogram of the mtTFA protein level in LCLs derived from healthy control and AGS patients. Β-Actin was used to normalize the total and the isolated cytoplasmic fraction and TOM20 was used to normalize the isolated mitochondrial fraction of the proteins. (B) Expression of mitochondrial MT-TL1 in healthy control and AGS patients (RNASEH2B- and RNASEH2A-mutated). B2M was used to normalize the gene expression. The bar graph represents the mean of three biological experiments. ANOVA test and post hoc analysis Tukey’s Test have been performed. * p < 0.05.