A9434

For murine chondrocyte experiments, chondrocytes were isolated from sterna of newborn pups (C57BL/6 J) age P1-P3 without consideration for sex. Cells were isolated by sequential digestion with pronase (2 mg/mL, PRON-RO, Roche) at 37°, followed by collagenase D (3 mg/mL, COLLD-RO, Roche) two times at 37°, and cultured in DMEM (Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (15140122, ThermoFisher, Waltham, MA, USA) and plated in tissue culture plates. For glutamine deprivation conditions, media was changed to high glucose DMEM containing glutamine or devoid of glutamine (Life Technologies, Carlsbad, CA, USA). For experiments, cells are treated with recombinant mouse IL-1β (211-11B, Peprotech, Cranbury, NJ, USA) at 10 ng/mL, CB-839 (S7655, Selleck, Chemicals, Houston, TX, USA), rapamycin (HY-10219, MedChem Express, Monmouth Junction, NJ, USA), ammonium chloride (A9434, Sigma-Aldrich, USA), L-asparagine (A0884, Sigma-Aldrich, St. Louis, MO, USA), or L-glutamatic acid (G1626, Sigma-Aldrich, St. Louis, MO, USA).

Enzymatic assays were performed using a modified protocol described previously^{37 (link),38 (link)}. Briefly, 2 µg of human recombinant GS obtained from Novus Biologicals (NBP2-52376) or purified GS obtained as described in the next section was added to the reaction mixture consisting of 100 mM imidazole/HCl (pH 7.2; I5513, Sigma-Aldrich), 50 mM glutamate (pH 7.2; G5889, Sigma-Aldrich), 20 mM ATP (pH 7.2; A7699, Sigma-Aldrich) and 20 mM MgCl₂ (M4880, Sigma-Aldrich). Unless otherwise indicated, 500 µl of reaction mixture was incubated for 2 min at 37 °C, and the reaction was initiated by adding NH₄Cl (A9434, Sigma-Aldrich), CH₃NH₂·HCl (M0505, Sigma-Aldrich), ¹⁵NH₄Cl (299251, Sigma-Aldrich) or ¹³C-methylamine (277630, Sigma-Aldrich) at the concentrations indicated in Fig. 4f,g or in the legends of Extended Data Fig. 3a,b. Aliquots (5 µl) of the reaction mixtures were sampled at the times specified in the figure legends, diluted 1:1,000 in LC–MS extraction solution and analyzed by LC–MS. K_m values were calculated in GraphPad 9.4 (Prism) by using a Michaelis–Menten equation. N²-Methyl-l-glutamine used in Fig. 2j was synthesized by replacing glutamate with 40 mM N-methyl-l-glutamate (ICN15555583, Fisher Scientific) in the reaction mixture described above. The reaction was incubated at 37 °C for 2 h, and an aliquot of the mixture was diluted 1:1,000 for LC–MS/MS analysis.