Chon 001

Human chondrocyte cell line (CHON-001) and 293T cells were purchased from ATCC (Manassas, VA, USA) and cultured in RPMI-1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% FBS (Thermo Fisher Scientific) and 2 mM glutamine (Sigma-Aldrich, St. Louis, MO, USA) at 37° C. CHON-001 cells were treated with IL-1β (10 ng/mL, Sigma-Aldrich) for 48 h to mimic OA in vitro as previously described [42 (link), 43 (link)].

The human chondrocyte cell line CHON-001 and mouse chondrocyte cell line ATDC5 were purchased from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). These cells were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco, Grand Island, New York) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, New York) and 100 U / mL penicillin and 100 μg / mL streptomycin (Hyclone, Logan, UT, USA). All cells were cultured in a humidified incubator containing 5% CO₂ at 37 °C. IL-1β (Sigma-Aldrich, Shanghai, China) was diluted to 10 ng / ml with serum-free medium, and CHON-001 and ATDC5 cells were stimulated with 10 ng/ml IL-1β to establish OA models. Subsequently, different concentrations of ICAR (Tauto Biotech, Shanghai, China) (0, 10, 20, 30 μM) were used to treat the cells. CYTOR overexpression plasmids and CYTOR short hairpin RNA (sh-CYTOR) were constructed by GeneChem (Shanghai, China). The transfection was performed with Lipofectamine® 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. 48 h later, the cells were harvested for subsequent experiments.

CHON-001, ATDC5, and HEK293 cells were purchased from the American Type Culture Collection (Manassas, VA, United States) or China Center for Type Culture Collection (Wuhan, China) and cultured in Dulbecco’s modified Eagle’s medium (12430104, Gibco, Grand Island, NY, United States) with 10% fetal bovine serum (12483020, Gibco, Grand Island, NY, United States), 100 U/ml penicillin, and 100 μg/ml streptomycin (15140122, Gibco, Grand Island, NY, United States) in 5% CO₂ at 37°C. CHON-001 and ATDC5 cell lines were stimulated with recombinant human IL-1β (SRP6169, Sigma-Aldrich, St. Louis, MO, United States) at concentrations of 1, 5, and 10 ng/ml, respectively, for 12 h to establish an in vitro model of OA.