Aconitase activity kit

To measure mitochondrial aconitase activity, an aconitase activity kit (Abcam, CA) was used as per manufacturer’s instructions. Briefly, MSCs in six‐well plates were exposed to normoxia or hyperoxia for 24 h. After treatment, cells were homogenized in 100 μL of assay buffer provided and spun at 20,000 g for 15 min at 4 °C to separate mitochondrial fractions. 300 μg of isolated mitochondria per sample were re‐suspended with 100 μL of assay buffer and then incubated for 1 h with the isocitrate substrate mixture at 25 °C and absorbance at 240 nm was recorded for 30 min using a SpectraMax™ reader. Catalytic activity of aconitase was determined by measuring the rate of formation of cis-aconitate as detected by increase in the absorbance^{65 (link)}.

Monoclonal Anti-actin antibody, anti-fatty acid synthase (FAS), Prussian Blue, sodium pantothenate and trypsin were purchased from Sigma Chemical Co. (St. Louis, MO). Anti-mitochondrial acyl carrier protein (mtACP), anti-aminoadipate semialdehyde dehydrogenase phosphopantetheinyl transferase (AASDHPPT), anti-NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 (NDUFA9), anti-NADH-ubiquinone oxidoreductase chain 1 (MTND1), DAPI and Hoechst 3342, were purchased from Invitrogen/Molecular Probes (Eugene, OR). NFS1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-PANK2, complex 1 activity kit, PDH activity kit and aconitase activity kit were purchased from Abcam (Cambridge, UK). Anti-mitochondrial 10-formyltetrahydrofolate dehydrogenase (ALDH1L2), anti-cytosolic 10-formyltetrahydrofolate dehydrogenase (ALDH1L1), anti-alpha-aminoadipic semialdehyde synthase, mitochondrial (AASS), anti-LYRM4, anti-Tau clone HT7, anti-ISD11, anti-PANK1 and anti-PANK3 were purchased from Thermo-Fisher (Waltham, MA). Anti-lipoic acid was acquired from Merck (Darmstadt, Germany). A cocktail of protease inhibitors (complete cocktail) was purchased from Boehringer Mannheim (Indianapolis, IN). The Immun Star HRP substrate kit was from Bio-Rad Laboratories Inc. (Hercules, CA).