Penicillin streptomycin 1x

GES-1 cells were donated by Dr. Dawid Kidane-Mulat (Austin, TX, USA) and cultured according to previous publications [31 (link)]. Cells were maintained in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (#SV30160.03, Hyclone-Cytiva, Pasching, Austria) and penicillin/streptomycin (1X) (#15140122, Thermo Fisher Scientific, Canada) at 37 °C in a humidified 5% CO₂ incubator. For the oxidative-stress-induced model, the GES-1 cells were treated with different concentrations of hydrogen peroxide (50, 100, 200, and 400 μM) for 3 and 24 h, respectively, at 37 °C. We selected 100 μM H₂O₂ for 3 h for our subsequent experiments.

Human knee joint tissues were obtained from OA patients during total knee arthroplasty as surgical waste under IRB approval of Duke Hospital (n = 6). Human articular cartilage from the tibial plateau and femoral condyle (both lesioned and non-lesioned) was finely diced and digested in pronase 0.1% (weight and volume, w/v, Roche, 10165921001) for 1 h, followed by 0.17% (w/v) type II collagenase (Sigma, C6885) in chondrocyte culture media for 16–18 h yielding a mean (± standard error of mean, SEM) 5.48 ± 1.01 × 10⁶ chondrocytes per gram of human articular cartilage [20 ]. Chondrocyte media contained DMEM/F-12, GlutaMAX™ (Thermo Fisher, 10565018) with 10% heat-inactivated fetal bovine serum (HI FBS, Thermo, 10082147), Penicillin–Streptomycin 1x (Thermo, 15140122), and 50 μg/ml l-ascorbic acid (Sigma-Aldrich, A8960). Isolated chondrocytes were seeded at a density of 200,000 cells/well in 24 well plates. The primary chondrocytes were cultured in a monolayer for 5 days in chondrocyte culture media and transduced with empty vector control, WT CBX4, or the mutant CBX4 in lentiviral constructs.

Bruce 4 (C57/B6 Strain) mouse embryonic stem cells (ESCs) and HEK293T were purchased from ATCC. Mouse Embryonic Fibroblasts (MEFs) were isolated as previously described [8 (link)]. Briefly, E13.5 C57BL/6 mouse embryos from 13.5 day-pregnant female mice were placed in (1X) PBS, the head and embryonic internal organs were dissociated from the abdominal cavity, then trypsinized and passed through a 10-ml syringe to produce single-cell suspensions, which were further expanded. MEFs and HEK293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) Sigma-Aldrich, Cat. No. D6429) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), Penicillin/Streptomycin (100 ug/ml; Thermo Fischer Scientific, Cat. No. 15140122), 2mM GlutaMAX (Gibco #,Cat. No. 35-050-061), and 0.1 mM Non-Essential Amino Acids 100X (Gibco, Cat. No. 11140050), at 37°C and 5% CO2. Bruce4 ESCs were cultured in complete ESC medium [Knock Out MediumDMEM (Gibco, Cat. No. 10829018), 20% ES-tested FBS (Pansera, Cat. No. P30-2602), Penicillin/Streptomycin 1X (Thermo Fischer Scientific, Cat. No. 15140122), GlutaMAX 1X (Gibco, Cat. No. 35-050-061), and Non-Essential Amino Acids 1X (Gibco, Cat. No. 11140050)], with 20 ng/mL mLIF (Santa Cruz Biotechnology, sc-4378).