Hexokinase

Respiration and ΔΨ were simultaneously determined using an Oxygraph-2k high resolution respirometer (Oroboros Instruments, Innsbruck, Austria) fitted with a potential sensitive tetraphenylphosphonium (TPP⁺) electrode. Mitochondria (0.05 mg/ml) were incubated at 37°C in 2 ml of ionic respiratory buffer (105 mM KCl, 10 mM NaCl, 5 mM Na₂HPO₄, 2 mM MgCl₂, 10 mM HEPES pH 7.2, 1 mM EGTA, 0.2% defatted BSA) with 5 U/ml hexokinase (Worthington Biochemical), and 5 mM 2-deoxyglucose. ADP was added sequentially to achieve the desired final concentrations with plateaus in respiration and potential achieved after each addition. A TPP⁺ standard curve was performed in each run by adding tetraphenylphosphonium chloride at concentrations of 0.25, 0.5, 0.75, and 1 μM prior to the addition of mitochondria to the chamber.

Differentiation was assayed by measuring creatine kinase levels using a modified method of Yun et al. (1997). One plate was collected every 24 h, the medium removed and wells rinsed with PBS, air dried for 15 min, and stored at −70°C until analysis. In brief, plates were thawed for 10–15 min and 500 μL of creatine kinase buffer (20 mmol/L glucose (Thermo Fisher Scientific), 10 mmol/L Mg acetate (Thermo Fisher Scientific), 1.0 mmol/L adenosine diphosphate (Sigma‐Aldrich), 10 mmol/L adenosine monophosphate (Sigma‐Aldrich), 20 mmol/L phosphocreatine (Calbiochem, San Diego, CA), 0.5 U/mL hexokinase (Worthington Biochemical, Lakewood, NJ), 1 U/mL of glucose‐6‐phosphate dehydrogenase (Worthington Biochemical), 0.4 mmol/L thio‐nicotinamide adenine dinucleotide (Oriental Yeast Co., Tokyo, Japan) and 1 mg/mL BSA, Sigma‐Aldrich) was added to each well including the standard curve wells containing creatine phosophokinase at concentrations between 0 and 40 mU/well. The optical density of each well at 405 nm was measured using a BioTek ELx800 (BioTek, Winooski, VT) plate reader. The differentiation assay was plated in two separate experiments with five replicate wells per experiment.