Atp measurement kit

ATP was measured using an ATP measurement kit (Molecular Probes, Carlsbad, USA) according to the manufacturer’s instructions. Briefly, cells were grown in 6-well plates to approximately 80% confluence. Approximately 1 × 10⁶ cells were washed with cold phosphate buffered saline (PBS) buffer, and then boiled in 100 μl Boiling Buffer (100 mM Tris, 4 mM EDTA, adjusted to pH 7.75 with acetic acid) for 90 s. Supernatants were retrieved by centrifugation at 10,000 × g for 1 min. ATP contents were determined by measuring the luminescence of supernatants mixed with Luciferase Assay buffer using a Varioskan™ Flash Multimode Reader (Thermo Scientific, Waltham, USA). ATP luminescence was normalized by protein concentration. To measure the generation of respiratory complex I-related ATP, a parallel set of cells was incubated with 200 nM rotenone (Sigma, St. Louis, USA) for 24 h before measuring the ATP.

Lentiviruses encoding mouse miR‐33a mimic, miR‐33a antisense oligonucleotide (ASO) or control vectors were purchased from ThermoFisher Scientific (Waltham, MA). Palmitic acid, bovine serum albumin, haematoxylin, eosin solution and oil red O stain kits were purchased from Sigma chemicals (Louis, MO, USA). Alanine transaminase (ALT), aspartate transaminase (AST) and triglyceride test kits were purchased from Wako (Richmond, VA). Anti‐total OXPHOS, anti‐α‐porin and anti‐NDUFA5 antibodies were purchased from Abcam (Cambridge, MA, USA); anti‐NFUFAF7 antibody was purchased from Sigma chemicals (Louis, MO); anti‐Tubulin was from Cell Signaling (Danvers, MA). ATP measurement kit was purchased from Molecular Probes (Molecular Probes, Carlsbad, USA).

B‐lymphocytes from several family individuals (II4, III3, III5, and III6) were immortalized as described previously (Hammerschmidt & Sugden, 1989). Individual oxidative phosphorylation system (OXPHOS) complexes were extracted from mitochondria using n‐dodecyl‐β‐d‐maltoside (Sigma, St Louis, MO, USA) with a detergent/protein ratio of 2.5 g/g. Proteins (60 μg) containing 0.5% Blue G‐250 (Sigma) and 5% glycerol were separated via electrophoresis on 3%–11% gradient blue native polyacrylamide gels as previously described (Xu et al., 2017).
ATP production rate was assessed using an ATP measurement kit (Molecular Probes, Carlsbad, CA, USA) according to the manufacturer's instructions. To measure mitochondrial ATP level, cells were incubated with 10 mM glucose (Sigma) or 5 mM 2‐DG with 5 mM pyruvate for 2 hr prior to the measurement. Fluorescence/luminescence was measured using a Varioskan^TM Flash Multimode Reader (Thermo Fisher Scientific, Waltham, MA, USA).

ATP was measured using an ATP measurement kit (Molecular Probes, Carlsbad, CA, USA) according to the manufacturer’s instructions. Complex I related ATP generatioin was measured by treating the cells with 200 nM rotenone for 24 h (Sigma). MMP was determined using the cationic fluorescent redistribution dye TMRM (Thermo Scientific) as described previously [44 (link)].

Mouse cardiac or primary mouse cardiomyocyte mitochondrial parameters, including ATP, endogenous basal oxygen consumption, citrate synthase activity, and complex I activity, were measured as previous reports (Nie et al., 2018a (link)). In brief, ATP levels were measured using an ATP measurement kit (Molecular Probes, Carlsbad, CA, United States), while endogenous basal oxygen consumption was measured with a Clark electrode in a water-jacketed chamber connected to a circulating water bath (Hansatech, Norfolk, United Kingdom). The citrate synthase activity was analyzed using a commercial measurement kit (Abcam). The activity of the electron transfer chain (ETC) complex I (NADH:ubiquinone reductase) was measured and expressed as a ratio to citrate synthase activity to account for mitochondrial enrichment.