Journal: Pharmacological Reports

Article Title: Targeting mitochondrial deubiquitinase USP30 to induce mitophagy in heteroplasmic mitochondrial diseases

doi: 10.1007/s43440-026-00829-7

Figure Lengend Snippet: Mutant cells exhibit reduced proliferation and ATP levels under OXPHOS-dependent conditions and resistance to mitochondrial inhibition under glycolytic conditions. 143B cybrid cells harboring 0% (N0), 40% (N40) or 80% (N80) of the m.8993T > G mtDNA mutation were cultured under glycolytic (glucose-containing medium) or OXPHOS-dependent conditions (galactose-containing medium – without glucose). (A) Confirmation of the mutant mtDNA load by ARMS-PCR. Data are mean ± SEM, n = 4 independent experiments (B) Mitophagy of N0 and N80 cells under glycolytic conditions was assessed by live imaging of the mitochondrial protein COX8 linked to both EGFP and mCherry: (i) schematic representation of the dual-fluorescent mitochondrial protein COX8-EGFP-mCherry: at neutral/alkaline pH, both EGFP and mCherry fluoresce, while at acidic pH (e.g. in mitolysosomes), only mCherry fluorescence is retained; (ii) representative images – red dots identify mitolysosomes (mCherry fluorescence only); (iii) quantification of the number of mitolysosomes per cell. n = 312–318 cells, from 3 independent experiments. Results are presented as median and interquartile range, with extremes presenting 10–90 percentiles, * p < 0.05, Mann-Whitney test. C) Cellular proliferation: growth over time after seeding in (i) glucose- or (ii) galactose-containing medium; (iii) representative images at 72 h after seeding. Initial cell density was similar across all conditions. Data are mean ± SEM, n = 5–7 independent experiments, non-linear regression following exponential growth equation. We used extra sum-of-squares F test for statistical comparison of growth curves: a single curve fits all data under glycolytic conditions (glucose), but not in OXPHOS-dependent conditions (galactose). D) ATP levels and ATP/ADP ratio quantified by HPLC of cells cultured in (i) glucose- and (ii) galactose-containing medium. Data are mean ± SEM, n = 3–4 independent experiments; * p < 0.05, one-way ANOVA followed by Dunnett’s multiple comparisons test. E) Resazurin metabolism after treatment of cells cultured in glucose- and galactose-containing medium with increasing concentrations of classical mitochondrial inhibitors for 24 h: (i) rotenone (complex I inhibitor); (ii) myxothiazol (complex III inhibitor); (iii) antimycin (complex III inhibitor); (iv) oligomycin (ATP synthase inhibitor). Data are mean ± SEM in % of N0 without drug treatment; n = 4 independent experiments

Article Snippet: Cells were seeded in glass-bottom Ibidi chamber slides (Ibidi, 80827) at a density of 15,000 cells/cm 2 , washed with DMEM (Gibco, 31966021), and incubated with a mixture of Lipofectamine LTX (Invitrogen, 15338030), Reagent plus and plasmid DNA (0.5 μg pCLBW-cox8-EGFP-mCherry, Addgene, 78520 [ ]) at a ratio of 2:1:1.

Techniques: Mutagenesis, Inhibition, Cell Culture, Imaging, Fluorescence, MANN-WHITNEY, Comparison

Journal: Pharmacological Reports

Article Title: Targeting mitochondrial deubiquitinase USP30 to induce mitophagy in heteroplasmic mitochondrial diseases

doi: 10.1007/s43440-026-00829-7

Figure Lengend Snippet: MF-094 promotes mitochondrial protein ubiquitination and formation of mitolysosomes. A) Quantification of MF-094 by UV-spectrophotometry: (i) UV spectrum of increasing concentrations of MF-094, showing an absorbance peak at 308 nm; (ii) calibration curve of MF-094: absorbance at 308 nm as a function of concentration; (iii) UV spectrum of 30 µM MF-094 after 24, 48, and 72 h of incubation under cell culture conditions in presence of cells; (iv) quantification of MF-094 in cell culture medium, with and without cells (initial concentration: 30 µM). Results from 3 independent experiments are presented as mean ± SEM. B) Metabolism of resazurin in cells treated with increasing concentrations of MF-094 under (i) glycolytic (glucose) or (ii) OXPHOS-dependent conditions (galactose); results from 3 to 4 independent experiments are expressed as mean ± SEM, as percentage of N0 cells without drug treatment. C) Mitochondrial protein ubiquitination was assessed by western-blot using an antibody against ubiquitin and a mitochondria-enriched extract from N80 cells treated with increasing concentrations of MF-094 under glycolytic conditions for 2 h: (i) representative blot and (ii) respective quantification. n = 6 independent experiments. D) TOM20 ubiquitination after N80 cell treatment with increasing concentrations of MF-094 under glycolytic conditions for 2 h: (i) representative blot – the panel identified with “high exposure” highlights ubiquitinated TOM20 (higher molecular weight than non-ubiquitinated TOM20); (ii) ubiquitinated TOM20 quantification. n = 6 independent experiments. C , D) Results are presented as mean ± SEM, * p < 0.05, one-way ANOVA with Dunnett’s multiple comparisons test. E) Mitophagy of N80 cells treated with 10 µM MF-094 under glycolytic conditions for 24 h was assessed by live imaging of the mitochondrial protein COX8 linked to both EGFP and mCherry: (i) representative images – red dots identify mitolysosomes (mCherry fluorescence only); quantification of the (ii) number and (iii) average area of mitolysosomes per cell. The inset graphs show amplified images of the regions of interest in the main graphs. n = 349–385 cells, from 4 independent experiments. Results are presented as median and interquartile range, with extremes presenting 10–90 percentiles, * p < 0.05, Mann-Whitney test

Article Snippet: Cells were seeded in glass-bottom Ibidi chamber slides (Ibidi, 80827) at a density of 15,000 cells/cm 2 , washed with DMEM (Gibco, 31966021), and incubated with a mixture of Lipofectamine LTX (Invitrogen, 15338030), Reagent plus and plasmid DNA (0.5 μg pCLBW-cox8-EGFP-mCherry, Addgene, 78520 [ ]) at a ratio of 2:1:1.

Techniques: Ubiquitin Proteomics, Spectrophotometry, Concentration Assay, Incubation, Cell Culture, Western Blot, Molecular Weight, Imaging, Fluorescence, Amplification, MANN-WHITNEY