15n2 l glutamine

The two non-Saccharomyces strains, Torulaspora delbrueckii (Biodiva, Lallemand) and Metschnikowia pulcherrima (Flavia, Lallemand), used in the study were propagated on yeast extract peptone dextrose (YPD) medium (2% glucose, 2% peptone, 1% yeast extract). Fermentations were carried out with a synthetic must (SM) that is similar to grape juice but with a defined composition, with modifications as in Bely et al. [24 (link)]. For the nitrogen source labelling experiment, the synthetic must contained 100 g/L glucose, 100 g/L fructose, and a mixture of 40% ammonium chloride and 60% amino acids as nitrogen sources (300 mg/L Yeast Assimilable Nitrogen, YAN). For the glucose labelling experiment, the SM contained 100 g/L glucose as the sole carbon source and ammonium chloride (300 mg/L YAN) as the nitrogen source. The concentration of organic acids, minerals, and vitamins in the SM were the same as those described by Su et al. [25 (link)]. SM was sterilised by filtration through 0.22 μm pore-size membrane filters (Labbox, Spain).
The labelled nitrogen sources, ¹⁵N ammonium chloride (99%), ¹⁵N₂ L-glutamine (98%), U-¹⁵N₄ L-arginine (98%), ¹³C₅ L-valine (97–98%), ¹³C₆ L-leucine (97–99%), and labelled ¹³C₆ glucose (99%) were obtained from Euriso-top (Cambridge Isotope Laboratories, Saint-Aubin, France).

In all, 2 × 10⁵ cells were plated onto 6-well plates (5 or 6 replicates for each cell type). The day after, the medium was replaced with fresh one containing the labeled isotopologue metabolite. For ¹³C₆ L-Leucine and ¹³C₆ L-Isoleucine (obtained from Cambridge Isotopes Laboratories) tracing experiment in Plasmax, cells were incubated for the indicated short time points or 43 h. For ¹⁵N L-Leucine and Isoleucine (Sigma Aldrich) tracing for 27 h. The labeling experiment with ¹⁵N L-Leucine in nutrient-deprived condition was conducted for 24 h in EBSS containing 2.5% FBS and 380 μM of ¹⁵N L-Leucine (Sigma Aldrich). For the ¹³C₅ L-Glutamine (obtained from Cambridge Isotopes Laboratories) tracing experiments, the cells were cultured in Plasmax with 0.65 mM of labeled compound in the presence of vehicle, GLSI (CB-839, 100 nM) for 23 h or ACLYI (BMS-303141, 10 μM) for 8 h. For tracing experiments with RPMI, cells were incubated with ¹³C₆ L-Leucine, ¹³C₅ L-Valine or ¹⁵N₂ L-glutamine (obtained from Cambridge Isotopes Laboratories or Sigma Aldrich) for 24 h. In the manuscript, we presented the labeling patterns derived from ¹³C₆ Leucine for KIC and C5-carnitine in ccRCC cells + VHL, while only the total pools from labeling experiments have been used, together with other steady state experiments, to measure the intracellular levels of aspartate, C3 and C5 carnitines.