Fig 1: Altered mitochondrial features in cybrid cells carrying MELAS, MERRF, m.5514 A > G (mt-tRNATrp), m.1643A > G (mt-tRNAVal), and m.14487 T > C (ND6) mutations. (A) Representative western blot of LONP1, AFG3L2 and CLPP peptidases in mutant and wild type (WT) cybrid cells. The membrane was also probed with porin as a loading control. Full-length western blots and lower-exposure blots of porin are included in supplementary information. (B) Densitometric analysis of LONP1, AFG3L2 and CLPP normalized to porin and represented as fold change relative to WT (top). Quantitative data are from at least three independent experiments. Results from this analysis are also shown as a heatmap (bottom). The color and the corresponding value in log2 scale are depicted on the left. (C) Representative Blue Native-PAGE of OXPHOS complexes in mutant and WT cybrid cells. Full-length blots and lower-exposure blots for those with high contrast are included in supplementary information. (D) Densitometric analysis of OXPHOS complexes normalized to complex-II (loading control) and represented as fold change relative to WT. (E) Cellular ATP determination in mutant and WT cybrid cells. (F and G) Determination of Ca2+ (F) and ROS (G) by flow cytometry in mutant and WT cybrid cells with Fluo-3 and MitoSOX Red, respectively. All data are the mean ± SEM of at least three different experiments. Differences from WT values were found to be statistically significant at *p < 0.05, **p < 0.01 and ***p < 0.001.
Fig 2: Failure to activate 40-kDa OMA1 at the IMM explains the absence of L-OPA1 cleavage in OXPHOS-dependent RPE1 cells. (A) Simultaneous L-OPA1 cleavage, 40-kDa OMA1 degradation and the generation of 60-kDa OMA1 in glycolytic RPE1 cells that stably expressed OMA1–HA and had been treated with CCCP. Lysates were immunoblotted with the indicated antibodies against OPA1 HA. (B) Impaired 40-kDa OMA1 degradation in OXPHOS-active RPE1 cells that stably expressed OMA1–HA (1 hour CCCP). (C) L-OPA1 is processed in the absence of detectable 60-kDa OMA1 in OXPHOS-active RPE1 cells that had been treated with oligomycin (OLG), as shown by immunoblotting. Glucose- and galactose-cultured RPE1 cells were incubated with 10 or 20 µM OLG for the times shown. (D) Protease protection assay. Mitochondria that had been isolated from control (DMSO) or CCCP-treated glycolytic RPE1 cells that stably expressed OMA1–HA were subjected to proteinase K (PK) treatment for the times indicated and then immunoblotted with antibodies against the indicated OMM, IMM and matrix resident proteins. (E) L-OPA1 processing is observed in the absence of detectable 60-kDa OMA1 in glycolytic RPE1 cells that had been treated with CCCP and cycloheximide (CHX) for 6 hours. (F–I) Silencing of AFG3L2 in OXPHOS-dependent RPE1 cells reveals a possible role for L-OPA1 cleavage in mitophagy. (F) Suppression of AFG3L2 expression enables L-OPA1 cleavage in CCCP-treated OXPHOS-dependent RPE1 cells. Immunoblotting for OPA1 reveals complex isoforms patterning. In galactose medium, band a (L-OPA1) is marginally decreased following treatment with AFG3L2 siRNA (note the appearance of band c), but is completely lost in the presence of CCCP (1 hour). (G) AFG3L2 siRNA triggers 40-kDa OMA1 degradation in OXPHOS-dependent RPE1 cells that stably expressed OMA1–HA. (H,I) Immunoblotting (top row) and densitometry-based quantification as a percentage of the untreated non-targeting siRNA control cells (bottom row) of (H) COXIV and (I) HSP60 levels as a measure of CCCP-induced mitophagy in AFG3L2-suppressed cells (20 hours) (means±s.d.; n = 3; one-way ANOVA, Tukey's post-test). <<, 60-kDa OMA1; <, 40-kDa OMA1; ns, not significant; NT, non-targeting; Tub, tubulin.
Fig 3: MTO1 defective cells exhibit proteostasis stress and an altered bioenergetic state. (A and B) Representative immunoblots showing the expression of CLPP, AFG3L2 and LONP1 in extracts of WT and MTO1 HF (A), and in MTO1 siRNA 1-, MTO1 siRNA 2- and Negative Control (NC) siRNA-transfected 143B cells (B). Porin was used as a loading control. Full-length blots are included in supplementary information (Fig. S19). The scatter plots show the densitometric measurements of the mitoproteases normalized to the loading control and represented as fold change relative to control cells. (C and D) Representative Blue Native-PAGE of OXPHOS complexes in WT and MTO1 HF (C), and in MTO1 siRNA 1-, MTO1 siRNA 2- and NC siRNA-transfected 143B cells (D). Full-length blots and lower-exposure blots of complex III are included in supplementary information (Fig. S20). The scatter plots show the densitometric measurements of OXPHOS complexes normalized to complex-II (loading control) and represented as fold change relative to control cells. (E) Analysis of oxygen consumption rate (OCR) of intact cells using different OXPHOS inhibitors. OCR was measured in each cell type under basal conditions and after sequential addition of oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (CCCP), rotenone and antimycin A. The scatter plot shows basal OCR (determined as the difference between OCR before oligomycin and OCR after rotenone/antimycin A), ATP-linked OCR (difference between OCR before and after oligomycin), proton leak (difference between basal OCR and ATP-linked OCR), reserve capacity (difference between the CCCP-stimulated rate and basal OCR), non-mitochondrial OCR (OCR after rotenone and antimycin A treatment), and maximal OCR (difference between OCR after CCCP and non-mitochondrial OCR). (F and G) Measurement of total and mitochondrial ATP levels in fibroblasts (F) and MTO1-silenced 143B cells (G). Cells were incubated with 5 mM glucose or 2.5 mM 2-deoxy-d-glucose plus 2.5 mM pyruvate (left and right, respectively) to determine total and mitochondrial ATP levels. Data are expressed as fold change respect to WT HF (F) or NC siRNA-transfected 143B cells (G) values. All data are the mean ± SD of at least three different experiments. Differences from control values were found to be statistically significant at *p < 0.05, **p < 0.01, ***p < 0.001. n.s.: non-significant differences.
Supplier Page from Abcam for Anti-AFG3L2 antibody