Pron ro

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).

Primary chondrocytes were harvested from sterna and ribs of P1-P3 mice using sequential digestion with pronase (2 mg·mL⁻¹, PRON-RO, Roche) at 37 degrees, collagenase D (3 mg·mL⁻¹, COLLD-RO, Roche) two times at 37 degrees, and cultured in DMEM (Life Technologies, USA) containing 10% FBS and 1% penicillin/streptomycin (Thermo Fisher). Primary chondrocytes are not passaged and experiments are completed within 5 days of isolation from mice. Primary macrophages (osteoclast precursors) were harvested from long bones of WT mice and cultured in M-CSF overnight before being added to chondrocyte cultures. Cells were incubated with recombinant IL-1β (10 ng·mL⁻¹), IKK2 inhibitor SC-514 (10 μmol·L⁻¹), BP (1 mg·mL⁻¹). Plat-E cells were used to generate retroviral particles.