Cb 839

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).

2, 2-dichloroacetophenone (DAP) was purchased from Sigma (St. Louis, MO). Stock solutions of the drug were prepared at 7 mM in dimethyl sulfoxide (DMSO). Stock solutions of CB-839 (Selleckchemicals, cat no: s7655) were prepared in DMSO at 87.95 mM. TTFA (2-Thenoyltrifluoroaceton, Cat# T27006), Rotenone (Cat# R8875), UPGL00004 (Cat#SML2472), 2-DG (Cat#D8375) and 3-Nitropropionic acid (Cat# N5636) were purchased from Sigma-Aldrich. Cells were treated from 24 h up to 3 days and then counted and analyzed by FACS.

CB-839 (S7655) was from Selleck Chemicals, bafilomycin A1 (88899-55-2), BPTES (19284) and compound 968 (17199) were from Cayman Chemical, and cycloheximide (C4859) and ISRIB (SML0843) were from Sigma-Aldrich. All chemical inhibitors were resuspended in DMSO. An equivalent amount of DMSO was added to control samples to control for any solvent-based effects.

Texas-red phalloidin was purchased from Invitrogen (Carlsbad, CA). CPI-613 and CB-839 were obtained from Selleckchem (Houston, TX). Antibodies against GLS1, GLUD1, HKI, HKII, PKM1, PKM2, PFKP, PDH, α-KGDH and p-PDHA1 (Ser293) were purchased from Cell Signaling Technology (Beverly, MA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an antibody that recognizes β-Actin were obtained from Sigma-Aldrich (St Louis, MO). For gene knockdown, the pLKO.1-puro TRC control shRNA targeting the gene encoding green fluorescent protein (GFP) and shRNAs targeting the GLS1 gene were purchased from Horizon Discovery (Waterbeach, UK).

10⁴ cells were seeded per well in 96-well plates in DMEM medium with 10% FCS. Carboplatin (ACCORD, 10 mg / ml) and Paclitaxel (KABI, 6 mg / ml), or Ironomycin (in-house drug), or CB-839 (at 10 μM, Selleckchem #S7655)), or Metformin (at 0.01 M, Sigma Aldrich #317240) were added the next day at the appropriate concentration. Cell viability was assayed for IC₅₀ determination at 48 hr for Carboplatin + Paclitaxel and at 72 hr for Ironomycin treatment or at 96 hr for the time course experiment by using the resazurin assay. To do so, 20 μL of resazurin reagent (0.05 mg / ml; Sigma Aldrich #R7017) was added to each well. Plates were incubated at 37°C for 2 hr and read in a Multi Detection plate reader (Fluostar, BMG Labtech).