Targeting Fatty Acid Oxidation Enhances Response to HER2-targeted Therapy
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Metabolomics Dataset for Figure(s) 2g, 3d and Supplementary Figure(s) 2c, 2d, 7i. File Name: Nandi et al_Nat Comms_2024_Metabolomics_Figure2g_3d_Supplementary_Figure2c_2d; Contains data for Figure 2g, Figure 3d and Supplementary Figure 2c and 2d File Name: Nandi et al_Nat Comms_2024_Metabolomics_Supplementary_Figure7i; Contains data for Supplementary Fig. 7i.
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Metabolomics and Gas Chromatography/Mass Spectrometry analysis: Metabolomics profiling, 13C-glucose and 13C-palmitate tracking analyses were performed as described (Andzejewski et al., 2014). Briefly, Cpt1a-proficient and -deficient NIC cells were cultures in unlabelled media in 6-cm dishes (Nunc) for 48 hours. Media was then changed to labeled media 25mM [U-13C]-glucose (Cambridge Isotope Laboratories, CLM-1396, 99% atom 13C) or [U-13C]-palmitate (Cambridge Isotope Laboratories, CLM-409, 99% atom 13C) for indicated time points. Cells were washed three times in ice-cold normal saline solution and water-soluble metabolites were extracted in 80% methanol. Samples dried by vacuum centrifugation (Lanconco) overnight at -1 °C were resuspended in 30 μl methoxyamine hydrochloride (MOX) in anhydrous pyridine and added to GC–MS autoinjector vials containing 70μl N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization reagent. The samples were incubated at 70 °C for 1 h, after which aliquots of 1 μl were injected for analysis. GC-MS data were collected on an Agilent 5975 C series GC/MSD system (Agilent Technologies) operating in election ionization mode (70 eV) for selected ion monitoring (SIM). The relative amount of each metabolite was determined from the integral ratios of the metabolites to the internal standard and normalized to the number of cells extracted using MassHunter software (Agilent Technologies) according to published protocols. Mass isotopomer distribution was determined using a published custom algorithm developed at McGill University (McGuirk).