15n 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.

Cells on six-well plates were washed twice with the wash buffer (75 mM ammonium carbonate, pH 7.4) and the plates were flash-frozen in liquid nitrogen. 800 μl extraction buffer (acetonitrile:methanol:H₂O = 4:4:2, −20°C) was added to the wells, scraped, and centrifuged by 21,000g for 20 min at 4°C. The supernatants were dried by vacuum centrifugation (Labogene) for 6 h at 20°C. Pellets were lysed in 50 mM Tris-KOH pH 8.0, 150 mM NaCl, 1% SDS, and used for protein quantification using the BCA assay (23225; Thermo Fisher Scientific). To measure steady-state levels of metabolites, the following internal standards were added to the extraction buffer: 2.5 mM amino acid standard (MSK-A2-1.2; CIL), 100 μg/ml citrate d₄ (485438; Sigma-Aldrich), 1 mg/ml ¹³C₁₀ ATP (710695; Sigma-Aldrich). No internal standard was added for the stable isotope-tracing experiments. Isotopologues used in the experiments are as follows: ¹³C₆ D-glucose (389374; Sigma-Aldrich), 2,3,3-²H₃ L-serine (DLM-582; CIL), ¹³C₆ L-leucine (605239; Sigma-Aldrich), ¹⁵N L-leucine (340960; sigma-Aldrich), ¹³C₅ L-valine (758159; Sigma-Aldrich), ¹⁵N L-valine (490172; Sigma-Aldrich). Isotopologues were added to the regular culture medium (MEM supplemented with 9.5% undialyzed FBS) and treated to cells as indicated in each figure legend.