13c6 l leucine

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.

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).