Oxygraph 2k system

Respiration measurements were performed in 2 ml of mitochondrial respiration medium 05 (MiR05) for each fiber bundle. O₂ consumption was measured using the high-resolution Oxygraph-2k system (Oroboros, Innsbruck, Austria) [25 , 26 ]. The results were normalized to the wet weight or CS activity of the permeabilized fiber bundles. All the experiments were performed at 37°C in a 2-ml chamber. Oxygen flux induced by the addition of cytochrome c (Cyt-c) increased no more than 10%, confirming the viability of the preparations during the experiments [26 ]. Multi-substrate titrations. This titration protocol was modified from previous protocols that have been described in detail [25 ]. All the titrations were performed in series as described below. The multi-substrate titration consisted of the sequential addition of G3P (10 mM); pyruvate, malate, and glutamate (PMG; 10, 10, and 20 mM, respectively); Succ (10 mM); adenosine diphosphate (ADP, 2.5 mM); Cyt-c (10 μM); oligomycin (Oligo, 2 μg/ml); carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP, 1 μM); rotenone (ROT, 0.5 μM); malonate (MALO, 10 mM); and cyanide (KCN, 5 mM). Evaluation of fatty acid oxidation: A second titration protocol was performed sequentially with G3P (10 mM; non-p-state) and ADP (2.5 mM; p-state). A third titration protocol involved palmitoyl-carnitine (PALM, 75 μM; non-p-state) and ADP (2.5 mM; p-state).

In order to measure mitochondrial dysfunction, high resolution respirometry was applied using Oxygraph-2K system (Oroboros Instruments, Innsbruck, Austria). Before the measurement, the cells were incubated with 50 µg/mL PM, 50 µg/mL PM, and 1 µM quercetin, or with 50 µg/mL PM and 3 µM quercetin for 24 h prior the experiment. After harvesting, HBE cells were centrifuged and resuspended in MEM at 1 × 10⁶ cells/cm³ concentration. Then the cells were added to the respiratory chambers to measure routine respiration. The procedure was performed according to previously described method [56 (link)]. Briefly, HBE cells were exposed to 4 µg/mL oligomycin, 1 µM FCCP, and 1 µM rotenone with 5 µM antimycin A to measure mitochondrial dysfunction in HBE cells. Non-phosphorylating leaks were determined with oligomycin (4 μg/mL). Maximum uncoupled electron transfer system (ETS) was measured using carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP; 1 μM), residual oxygen consumption (ROX) was determined by addition of rotenone (2.5 μM) and antimycin A (0.5 μM).

Oxygen consumption rate in intact cells suspension was measured by the Oxygraph-2k system (Oroboros Instruments, Innsbruck, Austria). DMSO or isoplumbagin-treated cells were detached from the plate by trypsinization and suspended at 5 × 10⁵ cells/mL in DMEM/KSFM medium supplemented with 10% fetal bovine serum into the O2k chambers. The oxygen consumption rate of DMSO or isoplumbagin treated cells samples were measured by adding 0.5 μM oligomycin, 0.5 μM FCCP, 1 μM rotenone, and 1 μM antimycin A. For cell permeabilization and measurement of respiration in permeabilized cells, 5 × 10⁵ cells suspended in 2 mL MiR05 buffer (110 mM D-sucrose, 0.5 mM EGTA, 3.0 mM MgCl₂, 60 mM lactobionic acid, 10 mM KH₂PO₄, 20 mM taurine, 20 mM HEPES, 1 g/L bovine serum albumin, and pH 7.1) were added in O2k chambers. After cells had stabilized at routine respiration, plasma membrane permeabilization was performed by adding 5 μM digitonin, and then, O2 consumption rate was measured in response to sequential additions of 10 mM glutamate and 2 mM malate or 10 mM succinate, followed by 5 mM ADP, 0.5 mM oligomycin, 0.5 μM FCCP, 1 μM rotenone, 1 μM antimycin A, 0.1 mM TMPD, 0.4 mM ascorbate, and 5 mM NaN₃.

The rate of mitochondrial respiration was monitored at 25°C using an Oxygraph-2k system (Oroboros) equipped with 2 chambers and DatLab software as previously described, with slight modifications [53 (link)]. In detail, 200–300 μg of heart mitochondria were added to 2 ml of a buffer containing 200 mM sucrose, 10 mM potassium phosphate, 0.1% bovine serum albumin, 10 mM Tris-HCl, 10 mM MgSO₄, and 2 mM EDTA, pH 7.0; and respiration was measured. Oxygen consumption was measured after the addition of the NADH-generating substrates malate (0.5 mM) and glutamate (0.5 mM). Then, ADP (0.15 mM) was added. To inhibit complex I activity, rotenone was added to a final concentration of 100 nM. Then, succinate (10 mM) was added, and complex II–dependent respiration was determined. Finally, KCN (2 mM) was added to inhibit complex IV activity.
Heart mitochondria from p27-deficient and wild-type littermates were always measured blinded in parallel, using the same conditions. The same setup was applied to measure respiration of heart mitochondria isolated from mice that had received caffeine with the drinking water or water. For each preparation, a second set of measurements was performed in a crossover design.