Journal: iScience

Article Title: Accumulated mtDNA mutations are linked to specific impairments in NADH-linked respiration

doi: 10.1016/j.isci.2026.115184

Figure Lengend Snippet: PolG Mut mitochondria possess selective impairments to NAD-linked Respiration (A) Schematic depiction of the substrates and inhibitors added during the OxPhos kinetics assay. Mitochondrial oxygen consumption ( J O 2 ) across OxPhos kinetics assay in mitochondria isolated from (B) BAT, (C) brain, (D) colon, (E) heart, (F) kidney, (G) Liver, (H) and lung tissue. Ratio of maximal complex I (CI) versus CII-supported mitochondrial J O 2 (I). N = 4–6 per group. Data are presented as mean ± SEM and analyzed using multiple unpaired t tests ( B–5H) or unpaired t test ( I), ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Substrates utilized are indicated as follows: creatine kinase (CK; 20 U/mL), ATP (5 mM), phospho-creatine (PCr; 1 mM), cytochrome c (Cyt C; 10 μM; pyruvate (Pyr; 5 mM), malate (Mal; 1 mM), octanoyl-carnitine (Oct; 0.2 mM), glutamate (Glut; 5 mM), rotenone (Rot; 0.5 μM), succinate (Succ; 5 mM) oligomycin (Oligo; 0.02 μM), malonate (Malo; 20 mM), calcium chloride (CaCl 2 ; 0.6 mM) glycerol-3-phosphate (G3P; 10 mM), and antimycin A (Ant A; 0.5 μM). Graphics were generated using BioRender.

Article Snippet: Following freeze fracture, 20μg of mitochondria were added to Respiration Buffer in the Oroboros O2k system followed by Cyt C (10μM).

Techniques: Isolation, Generated

Journal: iScience

Article Title: Accumulated mtDNA mutations are linked to specific impairments in NADH-linked respiration

doi: 10.1016/j.isci.2026.115184

Figure Lengend Snippet: Respiratory capacity within the electron transport system remains intact in PolG Mut mice (A–H) Schematic representing the maximal respiratory capacity protocol. Real-time oxygen consumption ( J O 2 ) in mitochondria isolated from (B) BAT, (C) brain, (D) colon, (E) heart, (F) kidney, (G) Liver, (H) and lung following serial titration of the mitochondrial uncoupling agent, carbonyl cyanide- p -trifluoromethoxyphenylhydrazone (FCCP, FC; 0.25 μM). (I) Maximal mitochondrial respiration achieved during FCCP titration. N = 5–6 per group, Data are presented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 depict significant post hoc LSD by two-way ANOVA; main effect PolG ( B–4H) or unpaired t test ( I). Substrates utilized are indicated as follows: pyruvate (Pyr; 5 mM), malate (Mal; 1 mM), octanoyl-carnitine (Oct; 0.2 mM), succinate (Succ; 5 mM) cytochrome c (Cyt C, 10 μM). Graphics were generated using BioRender.

Article Snippet: Following freeze fracture, 20μg of mitochondria were added to Respiration Buffer in the Oroboros O2k system followed by Cyt C (10μM).

Techniques: Isolation, Titration, Generated

Journal: iScience

Article Title: Accumulated mtDNA mutations are linked to specific impairments in NADH-linked respiration

doi: 10.1016/j.isci.2026.115184

Figure Lengend Snippet: Mitochondrial respiratory phenotypes are maintained in permeabilized tissue (A) Tissue slices were permeabilized in saponin before assessment of oxygen consumption ( J O 2 ). (B) Intact colon, (C) heart, and (D) liver J O 2 under multiple substrate conditions. (E) Ratio of complex I (CI) versus CII-supported respiration. (F) Dose-response to FCCP titration and (G) maximal respiratory capacity in intact bone marrow-derived mononuclear cells (BMMCs). (H) Mitochondrial J O 2 during OxPhos kinetics technique in permeabilized BMMCs. (I) Ratio of CI versus CII-supported respiration in permeabilized BMMCs. N = 5 per group. Data are presented as mean ± SEM, p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001,∗∗∗∗ p < 0.00001 depict significant multiple unpaired t tests ( A–6C and 6G), and unpaired t test ( D–6F and 6H). Substrates utilized are indicated as follows: creatine kinase (CK; 20 U/mL), ATP (5 mM), phospho-creatine (PCr; 1 mM), cytochrome c (Cyt C; 10 μM), pyruvate (Pyr; 5 mM), malate (Mal; 1 mM), octanoyl-carnitine (Oct; 0.2 mM), glutamate (Glut; 5 mM), rotenone (Rot; 0.5 μM), succinate (Succ; 5 mM) oligomycin (Oligo; 0.02 μM), malonate (Malo; 20 mM), calcium chloride (CaCl 2 ; 0.6 mM) glycerol-3-phosphate (G3P; 10 mM) antimycin A (Ant A; 0.5 μM), and carbonyl cyanide- p -trifluoromethoxyphenylhydrazone (FCCP, FC; 0.25 μM). Graphics were generated using BioRender.

Article Snippet: Following freeze fracture, 20μg of mitochondria were added to Respiration Buffer in the Oroboros O2k system followed by Cyt C (10μM).

Techniques: Titration, Derivative Assay, Generated

Journal: iScience

Article Title: Reversible dissociation of mitochondrial Complex V balances anabolic and energy-generating needs in cancer

doi: 10.1016/j.isci.2026.114889

Figure Lengend Snippet: Metabolic properties of BY1 and BY3 cells are altered by F o -F 1 dissociation in ways that support high levels of TCA cycle and ETC function (A) Left panel: free F o increases proton leak. The indicated cell lines, harvested in log-phase growth, were suspended in MiR05 respiration buffer, permeabilized with digitonin and sequentially exposed to cytochrome c , malate, pyruvate, glutamate, succinate, octanoylcarnitine, and glycerophosphate, The OCR at this stage, measured on an Oroboros respirometer, was designated as leak respiration, given that, in the absence of added ADP, no ATP was being produced. ADP was then added to initiate OXPHOS, followed by stepwise titration of carbonyl cyanide chlorophenylhydrazone (m-Cl-CCP) to promiscuously dissipate the proton gradient and achieve maximal uncoupled respiration, thus allowing absolute proton leak to be quantified. Right panel: Oligomycin blocks a greater fraction of the proton leak in BY3-TALED cells. OCRs of BY3 and BY3-TALED cells were measured as described above in the absence of ADP and m-CI-CCP and following the stepwise addition of oligomycin to block proton flow through F o . Each point represents the mean of 3–6 replicas ±1 SE. Statistical significance was assessed using a two-tailed Student’s t test. (B) ΔΨm is lower in BY1 than in BY3 cells. Cells in log-phase growth were stained with TMRM, and NAO average perk fluorescence was assessed by flow cytometry. (C) ATP half-lives are shortened by CV dissociation. The indicated cells, each stably expressing Cyto- or Mito-targeted iATPSnFR2HaloTag ATP sensors (leftmost and middle panels, respectively), were re-suspended in ice-cold MiR05 buffer (Oroboros, Inc.). After equilibrating to room temperature for 20 min, 2-DG and oligomycin were added to a final concentrations of 100 mM and 2.5 μM, respectively, and continuous flow cytometric analysis was performed to quantify t 1/2 ’s. In other experiments (rightmost panel), BY3 and BY3-TALED cells were permeablized with digitonin, and the ANT inhibitor carboxyatractyloside was added to block the export of ATP from the mitochondria and to allow for an assessment of the true intra-mitochondrial ATP t 1/2 . (D) Basal ATP levels are largely maintained in the presence of CV dissociation. The indicated cells, each stably expressing Mito- or Cyto-targeted iATPSnFR2HaloTag ATP, were grown under the indicated conditions, stained with Janelia Fluor JFX650 HaloTag Ligand and subjected to moving average continuous flow cytometry. (E) OCRs are altered by changes in F o -F 1 association. The indicated digitonin-permeabilized cells were exposed to pyruvate, malate, glutamate, succinate, and ADP. OCRs were quantified with an Orboboros respirometer. (F) FAO in WT-BY1 cells is suppressed by enforcing MT-ATP6-c expression. FAO was quantified by measuring the release of 14 CO 2 from 14 C-labeled palmitate. (G) Mitochondrial matrix pH of cells with increased free F o is maintained in the normal range. The indicated cell lines, stably expressing Mito- or Cyto-targeted pSypHER GFP, were subjected to flow cytometry during log-phase growth. (H) The mitochondrial matrix of cells with free F 1 tends to remain relatively oxidized. The indicated cell lines stably expressing Cyto- or Mito-targeted roGFP were subjected to flow cytometry during log-phase growth. Data are shown as individual data points with the mean ± SD indicated. Statistical significance was assessed using ordinary one-way ANOVA. p values are indicated as follows: ∗ p < 0.05,∗∗ p < 0.01, ∗∗∗ p < 0.001,∗∗∗∗ p < 0.0001.

Article Snippet: To measure mitochondrial ATP half-life, 2–3 x 10 6 cells stably expressing the above-described Mito-targeted iATPSnFR2HaloTag ATP sensor were harvested by trypsinization and resuspended in 1 mL of Mir05 respiration buffer that contained no glucose (Oroboros, Inc.).

Techniques: Produced, Titration, Blocking Assay, Two Tailed Test, Staining, Fluorescence, Flow Cytometry, Stable Transfection, Expressing, Labeling