13c5 15n2 labeled l glutamine

Cells were seeded at 3 × 10⁵/ml (replicates of 3) and treated with vehicle (DMSO) or CB-839 at 500 nM for 8 h, followed by incubation in glutamine-free RPMI 1640 supplemented with ¹³C₅,¹⁵N₂-labeled L-glutamine at 300 mg/L (Cambridge Isotope Laboratories) for up to 12 h, in the presence of vehicle or drug. Flash frozen cell pellets (~ 1 × 10⁶ cells) or supernatants (50 μl) were extracted and subjected to analysis by ultra-high pressure liquid chromatography and mass spectrometry (UHPLC/MS) as was previously described (16 (link)). Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of ¹³C and ¹⁵N isotopes were performed using MAVEN (Princeton University, Princeton, NJ) and manually validated.

Cells were seeded at 3 × 10⁵/mL (replicates of three) and treated with vehicle (dimethylsulfoxide) or drug (AC220 or CB-839) for 8 hours, followed by incubation in regular RPMI 1640 (for unlabeled samples) or glutamine-free RPMI 1640 supplemented with ¹³C₅,¹⁵N₂-labeled L-glutamine (Cambridge Isotope Laboratories) for up to 12 hours in the presence of vehicle or drug. Flash-frozen cell pellets (~5 × 10⁵ cells) or supernatants (50 µL) were extracted and subjected to analysis by ultra-high-performance liquid chromatography and mass spectrometry as described previously [13 (link)]. Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of ¹³C and ¹⁵N isotopes were performed using MAVEN (Princeton University, Princeton, NJ) and validated manually. Extracellular flux analysis of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was performed using the Seahorse XF24 Analyzer as described previously [12 (link)] except that the assay medium contained 2 g/L glucose and 2 mmol/L glutamine.