Targeting Fatty Acid Oxidation Enhances Response to HER2-targeted Therapy
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Data Published in Manuscript, DOI: 10.1038/s41467-024-50998-3 Metabolomics Dataset for Figure(s) 2g, 3d and Supplementary Figure(s) 3c, 3d, 7i. File Name: Nandi et al_Nat Comms_2024_Metabolomics_Figure2g_3d_Supplementary_Figure3c_3d; Contains data for Figure 2g, Figure 3d and Supplementary Figure 3c and 3d 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 Metabolic profiling and isotope tracing analyses using 13C-glucose and 13C-palmitate were performed at the Metabolomics Innovation Resource, McGill University. Briefly, Cpt1a-proficient and -deficient NIC cells (n = 3 cell lines per genotype, analyzed in triplicate) were cultured in unlabelled DMEM supplemented with 2% dialyzed FBS (Wisent, 080-910) in 6-cm dishes (Nunc) for 48 hours. The media was then replaced with glucose-free DMEM supplemented with 2% dialyzed FBS and 25mM [U-13C]-glucose (Cambridge Isotope Laboratories, CLM-1396, 99% atom 13C) for either 30 minutes or 2 hours, or [U-13C]-palmitate (Cambridge Isotope Laboratories, CLM-409, 99% atom 13C) for 24 hours. Additionally, some dishes were kept in unlabeled media as controls. Cells were washed three times in ice-cold saline solution (NaCl, 0.9g/L), and water-soluble metabolites were extracted in 80% methanol (GC/MS grade). After two 10-minute rounds of sonication (30 seconds on/ 30 seconds off at high intensity) on slurry ice using the Bioruptor UCD-200 sonicator, the homogenates were centrifuged at 15,000 × g for 10 minutes at 4C. Supernatants were collected and an internal standard, 800 ng Myristic acid-D27, was added to each sample. Samples dried by vacuum centrifugation (CentriVap Concentrator; Labconco, KS, USA) overnight at -1 °C were resuspended in 30 μl of 10 mg/mL methoxyamine hydrochloride in anhydrous pyridine and incubated for 30 minutes at room temperature. Samples were then transferred to GC–MS autoinjector vials containing 70μl N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) derivatization reagent and incubated at 70 °C for 1 hour. A blank sample, composed of 30 μl of 10 mg/mL methoxyamine-HCl pyridine and 70μl of MTBSTFA, was also prepared. A volume of 1 μl of sample was injected splitless with an inlet temperature of 280 °C into the GC-MS instrument, Agilent 5975C. Metabolites were resolved by separation on a DB-5MS + DG (30m x 250 μm x 0.25 μm) capillary column (Agilent, CA, USA). Helium was used as the carrier gas with a flow rate such that Myristic-D27 acid eluted at approximately 18 minutes. Metabolites were ionized by electron impact at 70 eV. All samples were injected using scan (50-1000m/z) and selected ion monitoring (SIM) mode. In all experiments, Cpt1a-proficient NIC cells were used as controls. The sample preparation and data collection order for biological and technical replicates was randomized. All metabolites described in this study were validated against authenticated standards to confirm mass spectra and retention times. The relative amount of each metabolite was determined from the integration of ion intensities and normalized to the number of cells extracted using MassHunter Quant software (v12.0.893.1, Agilent) according to published protocols. Mass isotopomer distribution analysis was determined using a custom in-house algorithm developed at McGill University.