Aconitase assay kit

We purchased tebufenpyrad (96% purity) from AK Scientific Inc. (Union City, CA), pyridaben (99.1% purity) from Chem Services (West Chester, PA), and rotenone (95–98% purity) and hydrogen peroxide (30 wt. % in H₂O) from Sigma (St. Louis, MO). DMSO was purchased from Fisher Scientific (Fair Law, NJ). We purchased RPMI 1640 media, fetal bovine serum (FBS), L-glutamine, penicillin, streptomycin and Sytox green nucleic acid fluorescence stain from Molecular Probes (Eugene, OR), the Muse® Count & Viability Assay Kit (Catalog # MCH100102) from EMD Millipore (Billerica, MA), and the 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) fluorescent probe and MitoTracker red CMXROS and MitoTracker green dyes from Invitrogen (Carlsbad, CA). The Cell Titer 96® AQueous Non-Radioactive Cell Proliferation assay kit and Cell Titer Glo Luminescent Cell Viability assay kit were bought from Promega (Madison, WI). The Aconitase assay kit was purchased from Abcam (Cambridge, MA). Oligomycin, hydrogen peroxide, carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (FCCP) and antimycin A were purchased from Sigma Aldrich (St. Louis, MO), and the Seahorse FluxPak calibration solution was bought from Seahorse Biosciences (Billerica, MA).

Aconitase activity assays were performed using the Aconitase Assay Kit (ab83459) purchased from Abcam. 293T cells were grown in the presence and absence of BafA1(10nM) and/or ferric ammonium citrate (0.1mg/ml) for 24 hours. 1× 10⁶ cells per well were processed according to manufacturer’s protocol and aconitase activity was read out as the increase in absorbance at 240nm read in 45 second intervals over 30 minutes at room temperature on a SpectraMax M3 plate reader (Molecular Devices). Absorption at values 240nm were normalized to protein concentration as determined by BCA (Pierce) and converted to moles of cis-aconitate formed (extinction coefficient: 2.2 OD mM⁻¹/well).