Metabolic Profiling of Isocitrate Dehydrogenase

143Bwt and 143Bcytb and UOK262 cells were cultured as described^{9 (link),15 (link)}. MEFs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS, Hyclone), penicillin/streptomycin and 6mM L-glutamine. Cell growth was monitored in sub-confluent cultures by trypsinizing and counting with a hemacytometer. Glucose, lactate, and glutamine were measured as described^{23 (link)}. Isotopic labeling was performed in DMEM with 10% dialyzed FBS supplemented with either 15mM D-[U-¹³C]glucose or 2mM L-[U-¹³C]glutamine. Aqueous metabolites were analyzed using an Agilent 6970 gas chromatograph and an Agilent 5973 mass selective detector. To determine relative metabolite abundance across samples, the area of the total ion current peak for the relevant metabolite was compared to that of an internal standard (2-oxobutyrate) and normalized for protein content. Analysis of ¹³C enrichment and mass isotopomer distribution was performed as published^{11 (link),24 (link)}. For radioisotope lipid synthesis assays, cells were cultured for 18 hours in complete medium supplemented with ³H₂O or ¹⁴C tracers (Perkin Elmer). Lipids were extracted and analyzed as described^{3 (link)}. For lipid synthesis experiments using NMR, cells were plated in 10-cm dishes and allowed to proliferate exponentially in medium containing D[U-¹³C]glucose and unlabeled glutamine, or unlabeled glucose and L[U-¹³C]glutamine until two confluent 15-cm dishes were obtained. Lipids were then extracted and analyzed by ¹³C NMR as described^{3 (link)}. Gene silencing was performed using commercial small interfering RNAs (Thermo) directed against IDH1, IDH2 or IDH3. Cells were transfected with DharmaFECT transfection reagent (Thermo) and protein abundance was examined after 72 hours using western blots with commercial antibodies (Santa Cruz Biotechnology). Cells were incubated with L[U-¹³C]glutamine for two hours prior to extraction of metabolites for mass isotopomer distribution analysis. Stable silencing used lentiviral shRNAs from the Mission shRNA pLKO.1-puro library (Sigma). Additional details are in the online Methods.