1 13c glucose

Media and chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Tracers were purchased from Cambridge Isotope Laboratories: [1-¹³C]glucose (99.6 atom% ¹³C), [1,2-¹³C]glucose (99.8%), [1,6-¹³C]glucose (99.2%), and [U-¹³C]glucose (99.3%). M9 minimal medium was used for all tracer experiments. All solutions were sterilized by filtration.

Chemicals and M9 minimal medium were purchased from Sigma-Aldrich (St. Louis, MO). Isotopic tracers were purchased from Cambridge Isotope Laboratories (Tewksbury, MA): [1,6-¹³C]glucose (99.2 % ¹³C), [1,2-¹³C]glucose (99.7 %), [1-¹³C]glucose (99.5 %), [2-¹³C]glucose (99.5 %), [3-¹³C]glucose (99.5 %), [4,5,6-¹³C]glucose (99.5 %), and [1,2-¹³C]xylose (99.2 %). The isotopic enrichment of all tracers and the composition of tracer mixtures used in parallel labeling experiments were validated by GC-MS analysis as described in (Sandberg et al., 2016 (link)) and (Cordova and Antoniewicz, 2016 (link)). SFM4CHO medium (GE Healthcare Life Sciences SH3054901) and DMEM medium (Corning 17-207-CV, without glucose, glutamine, and sodium pyruvate) were purchased from Fisher Scientific (Pittsburgh, PA).

For ¹³C labeling experiments, semiconfluent cells in a 10-cm dish were cultured in glucose-free DMEM (Sigma) with the compounds listed in Supplementary Table S2 and a 25 mmol/L 1:1 mixture of (U-¹³C₆)glucose and (1-¹³C])glucose (Cambridge Isotope Laboratories) overnight. Cells were quenched with 1 mL ice-cold methanol and collected with a cell scraper. Four volumes of chloroform were added, and the cells were lightly vortexed and chilled in ice for 30 minutes for deproteinization. After centrifugation, the aqueous phase was collected and evaporated at room temperature. Dried polar metabolites were dissolved in 50 μL of 2% methoxyamine hydrochloride in pyridine (Pierce), sonicated for 30 minutes, and held at 37°C for 3 hours. After dissolution and reaction, 90-μL MBTSTFA + 1% TBDMCS (Pierce) was added and samples were incubated at 55°C for 1 hour. Gas chromatography/mass spectrometry (GC/MS) analysis was performed using an Agilent 6890 GC equipped with a 30-m DB-35MS capillary column connected to an Agilent 5975B MS operating under electron impact (EI) ionization at 70 eV. The identity and values of metabolites in glycolysis and OXPHOS were quantified and measured at the SickKids SPARC BioCentre in Toronto, Canada.

Culture materials were purchased from Cellgro (Mediatech, Manassas, VA). Chemicals were purchased from Sigma-Aldrich (St. Louis, MO). [1-¹³C]Glucose (99.5 atom% ¹³C), [2-¹³C]glucose (99.5%), [3-¹³C]glucose (99.5%), and [4,5,6-¹³C]glucose (99.8%) were purchased from Cambridge Isotope Laboratories (Andover, MA). Glucose stock solutions were prepared at 250 g/L in phosphate buffer saline (PBS). For experiments involving tracer mixtures, new glucose stock solutions were prepared by mixing the appropriate stock solutions at the desired ratio. The growth medium was Dulbecco’s modified Eagle medium (DMEM, Cat. No. 10-013-CV) supplemented with 10% fetal bovine serum (FBS, Cat. No. 35-011-CV) and 1% penicillin-streptomycin solution (PS, Cat No. 30-004-CI).