Rotenone antimycin a

The oxygen consumption rate (OCR) was measured with a Seahorse XF extracellular flux analyzer according to the manufacturer’s instructions (Agilent, Santa Clara, CA, USA). Primary fibroblasts were seeded at 8 × 10⁴ cells per well in a specialized 24-wells microplate and incubated at 37 °C in 5% CO₂ overnight. Culture medium was replaced with XF assay medium supplemented with 10 mM glucose (103577-100, Agilent), 1 mM pyruvate (103578-100, Agilent) and 2 mM glutamine (103579-100, Agilent) and cells were incubated in the absence of CO₂ for 45 min before measurement. OCR was determined before injection of specific metabolic inhibitors and after successively adding 1.5 μM oligomycin, 1 μM FCCP, and 0.5 µM rotenone/antimycin A (Sigma-Aldrich). OCR data were normalized according to fluorescent cell counting using BioTek Cytation (BioTek, Winooski, VT, USA). Wave software was used to analyse Seahorse measurements.

Mitochondria were isolated from snap‐frozen quadriceps by Mitocheck Mitochondrial Isolation kit (Cayman chemical 701010). ATP content was quantified in 20 μg of fresh isolated mitochondria by CellTiter‐Glo^® Luminescent Cell Viability Assay (Promega G7570). Aconitase activity was measured in quadriceps homogenates by enzymatic assay (Cayman Chemical 705502). Oxygen Consumption Rate (OCR) measurements were conducted using a Seahorse XFe96 analyzer according to manufacturer’s protocol. C2C12 cells were directly differentiated in XFe96 cell culture plates and treated with 10% C26 CM, ferric citrate 250 nM, or the combination of both for 48 h and incubated in 5% CO₂ at 37°C. One hour prior to analysis, growth medium was replaced with assay medium (DMEM without phenol red and sodium bicarbonate (Corning 90‐013‐PB) that was supplemented with 1 mM of pyruvate, 2 mM of l‐glutamine, and 10 mM of glucose, pH 7.4) and incubated in a non‐CO₂ incubator. During assay, 1 μM of oligomycin (Sigma 495455), 1 μM of FCCP (Sigma C2920), and 0.5 μM of rotenone/antimycin A (Sigma R8875 and A8674) were sequentially injected into each well in accordance with standard protocols. Absolute rates (p moles/min) were normalized to μg of protein determined by Bradford Assay (BioRad 5000006).

Measurement of OCR and ECAR were performed in XF96 plates with XF Extracellular Flux Analyzer (Seahorse Bioscience). Fibroblasts were seeded in collagen (BD Biosciences) coated XF 96-well plates (Seahorse/Agilent) in octuplicates at 1.2x10⁴ cells/well in 100 μl of growth medium. Mitochondrial activity was evaluated using the Seahorse XF Cell Mito stress Test Kit, according to manufacturer’s instructions (Agilent). In the metformin treatment experiments, cells were plated at 5x10³ cells/well 24–28 hours prior to the assay. Oligomycin (75351, Sigma-Aldrich), FCCP (C2920, Sigma-Aldrich), and Rotenone/Antimycin A (R8875 and A8674, Sigma-Aldrich) were used at 1.5 μM concentration, after a titration experiment. Glycolytic activity was evaluated using the XF Glycolysis Stress Test according to manufacturer’s instructions (Agilent). Glucose (G8769, Sigma-Aldrich) was used at 10 mM, Oligomycin at 1 μM and 2-D-Deoxy-Glucose at 50 mM (D6134, Sigma-Aldrich). Cell content was normalized using crystal violet. The post-normalization values of OCR and ECAR reflect both the metabolic activities of the cells and the number of cells being measured. Data were further processed according to manufacturer’s instructions.

Oxygen consumption was measured on a Seahorse XF24 analyser at 37°C. Seahorse XFe24 FluxPak mini (102342-100) and Seahorse XF base medium (102353-100) was purchased from Agilent (California). Oligomycin, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and Rotenone/Antimycin-A were purchased from Sigma. Forty-eight hours before the assay, 40,000 siSRSF3 or siNeg HepG2 cells were seeded in a Seahorse XF24 analyser plate. After inducing enough lipid droplets by pretreating HepG2 cells with 0.2 mM PA for 24 h, we replaced the medium with Seahorse XF base medium and measured the mitochondrial respiration in the basal state and after 1 μM Oligomycin, 2 μM FCCP, and 0.5 μM Rotenone/Antimycin-A treatment. Further, in another assay, instead of pretreating cells with PA to produce lipid droplets, we added excessive exogenous PA and then measured oxygen consumption. Adenosine triphosphate (ATP) production, basal respiration, maximal respiration, and spare respiratory capacity were calculated, respectively. Cells were lysed and protein concentration was taken for normalization.

The effect of LIPH silencing on mitochondrial respiration in MDA‐MB‐231 cells was determined using the Seahorse assay in an XF24 extracellular flux analyser (Seahorse Bioscience, North Billerica, MA, USA) in accordance with the manufacturer's instructions. Briefly, control and LIPH‐silenced MDA‐MB‐231 cells were cultured overnight in XF24 (V7) microplates (2 × 10⁴ cells/well). The cells were cultured in basic medium containing glucose, glutamine, and sodium pyruvate at 37°C in a non‐CO₂ incubator for 1 hour and the basal oxygen consumption rates (OCR) of individual wells of cells were measured thrice at 5 minutes intervals. Subsequently, cells were sequentially treated with rotenone/antimycin A (Krebs cycle inhibitors, all from Sigma) to quantify ATP production, proton leak, maximal respiration, and spare respiratory capacity after each treatment using the equipped software, normalized to protein levels measured via the BCA assay.

The OCR was measured in A375VR cells using a Seahorse XFp extracellular flux analyzer (Seahorse Bioscience). In brief, A375VR cells were seeded at a density of 10,000 cells per well on a Seahorse XFp plate in DMEM medium. The next day, the medium was replaced with Seahorse XF DMEM medium pH 7.4. Then, cells were incubated for 45 min at 37 °C and 0% CO₂ and treated with either 20 µM ETO (Merck) or RANO at the indicated concentrations during the last 15 min. Basal levels of OCR and ECAR were then recorded, followed by sequential injections of 1 µM oligomycin (Sigma), 1 µM FCCP (Sigma), 0.5 µM rotenone/antimycin A (Sigma) and 125 mM 2-deoxy-d-glucose (Sigma). OCR and ECAR data were normalized to the protein content as assessed by Bradford assay (Bio-Rad).

One day prior to the assay, CD8 T cells were cultured in substrate-limited growth medium (Seahorse XF DMEM [pH 7.4] medium [Agilent] supplemented with 0.5 mM glucose, 1 mM glutamine, 1% fetal bovine serum, 0.5 mM L-carnitine, and 5 U/mL human IL-2). On the day of the assay, cells were washed and resuspended in Seahorse XF DMEM medium supplemented with 2 mM glucose and 0.5 mM L-carnitine. Cells (10⁵ cells/well) were then plated onto Cell-Tak-coated Seahorse cell plates. The OCR was measured in response to Omy (1.5 μM), carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 1.5 μM) and rotenone/antimycin A (0.1 μM and 1 μM, respectively) (all from Sigma-Aldrich). Measurements were taken using a Seahorse XFp analyzer (Agilent).