Chon 001

Human chondrocyte cell line CHON-001 (ATCC CRL-2846) was obtained from ATCC. Cells were cultured in ATCC-formulated Dulbecco’s modified Eagle’s medium (DMEM, cat. no. 30–2002) supplemented with 0.1 mg/ml G-418 and 10% heat-inactivated fetal bovine serum in an incubator (37 °C, 5% CO₂). Cells were harvested during the logarithmic phase for subsequent experiments.

CHON-001 cells were purchased from ATCC (Cat No. CRL-2846) and cultured according to instructions. Cells were grown in a complete DMEM medium enhanced with 1% P/S and 10% FBS and incubated in a 5% CO₂ incubator at 37°C. Cells were grown up to 80% confluence and each experiment was performed after three passages.

Human chondrocytes cell line CHON-001 (ATCC, Rockville, MD) were maintained in DMEM (Thermo, Waltham, MA) with 10% FBS (Thermo Fisher Scientific, USA) under a 5% CO₂ atmosphere with 95% humidity [15 (link)]. We obtained the cell treatment reagents human TNF-α and Nintedanib from Sigma-Aldrich (St. Louis, USA). CHON-001 cells were stimulated with TNF-α (10 ng/mL) with or without the presence of Nintedanib (15 μM) for 24 h. For the PKA inhibitor experiment, the cells were treated with PKA inhibitor H89 (10 μM) for 24 h.

The immortalized HAC cell line (CHON-001; CRL-2846; ATCC, Manassas, VA, USA) was cultured in DMEM (M22650; R&D systems) containing 0.1 mg/ml G418 disulfate salt (4131; R&D systems) and 10% FBS (R&D systems). HAC cells were used to transiently transfect with exogenous nucleotides or vectors using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) for 36 h. The oligonucleotides were circ_0045714-siRNA (si-circ_0045714), PIK3R3-siRNA (si-PIK3R3), negative control (NC)-siRNA (si-NC), miR-331-3p mimic, miR-NC mimic, miR-331-3p inhibitor (anti-miR-331-3p), and miR-NC inhibitor (anti-miR-NC). The vectors were empty pCD5-ciR vector (GENESEED, Guangzhou, China), recombinant pCD5-ciR-circ_0045714 vector, pmiR-Reporter vector (Promega, Madison, WI, USA) expressing circ_0045714 containing the wild type (WT) or mutant type (MUT) of miR-331-3p response elements, and pmiR-Reporter vector (Promega, Madison, WI, USA) expressing PIK3R3 3’UTR containing WT or MUT of miR-331-3p response elements. Single and co-transfection models were performed per the instructions, and transfected cells at 36 h were harvested for further assays.

The human chondrogenic cell line CHON-001 and HEK293T cells were bought from ATCC and maintained in DMEM (Gibco, CA, USA) supplemented with 10% FBS (vol/vol) (Gibco, cat no. 10100147), 100 μg/ml penicillin/streptomycin (Gibco, cat no. 15140148). The cells were cultured in a humidified environment at 37 °C and 5% CO₂. The CHON-001 cells were exposed to indicated concentration (10 ng/ml) IL-1β (Merck, cat no. SRP6169) for 12 h to establish OA model.