Insulin transferrin selenium its g

All protocols were approved by the institutional review board of Seoul St. Mary’s Hospital (KC21SISI0337) and were performed in accordance with the Declaration of Helsinki. Human articular cartilage was acquired from patients for replacement arthroplasty or joint replacement surgery, and the chondrocytes were obtained from the cartilage and maintained in DMEM (WELGENE) containing 10% FBS (Corning), 100 units/mL of penicillin, and 100 mg/mL of streptomycin (Invitrogen), and cultured at 37°C in 5% CO₂ humidified incubator. Human OA chondrocytes were seeded at 3×10⁵ cells/well or 2×10⁵ cells/well into 6-well or 12-well plates, respectively. Following 24 h of starvation with insulin-transferrin-selenium (ITS-G, Thermo Fisher, Waltham, MA, USA), cells were stimulated with 10 ng/ml IL-1β with or without various concentrations of TA (0.5, 1, or 2 μM), recombinant human IL-1R1 Fc (1 μg/ml, Abcam), or anti-IL-1β neutralizing antibody (1 μg/ml, Abcam) in serum-free DMEM for 1, 3, or 48 h. recombinant human IL-1R1 Fc or anti-IL-1β neutralizing antibody were used as a positive control and medium only group was used as a negative control. The supernatant and cells were collected for further analysis.

Dulbecco’s Modified Eagle Medium (DMEM), RPMI1640, DMEM/F12, phosphate-buffered saline (PBS), penicillin–streptomycin 100X, 0.25% trypsin, 0.05% trypsin, fetal bovine serum (FBS), exosome-depleted FBS, minimum-essential amino acids, sodium pyruvate, L-glutamine, epidermal growth factor (EGF), and insulin–transferrin–selenium (ITS-G) were purchased from Gibco (ThermoFisher Scientific, Waltham, MA, USA); adenine, hydrocortisone, and high cholera toxin from MilliporeSigma (Burlington, MA, USA); and rock inhibitor and primocin from Cayman Chemicals (Ann Arbor, MI, USA) and InvivoGen (San Diego, CA, USA), respectively.

NALM-6 isogenic knock-in cell lines including different hotspot mutations in the HEAT domain of SF3B1 (parental, K700E, K666N, and H662Q) were from a previous study (11 (link)) and mutations were confirmed by Sanger sequencing. UM cell lines 92.1 (SF3B1wt) and Mel202 (SF3B1mut) were acquired from Martine de Jager (department of ophthalmology LUMC, The Netherlands). Pancreas carcinoma (PDA) cell line panc1 (SF3B1wt) was obtained from the LEXOR group (Amsterdam UMC, The Netherlands) and panc05.04 (SF3B1mut) was directly bought from ATCC and CLL cell lines PGA (SF3B1wt) and CII (SF3B1mut) were a kind gift from Tanja Stankovic (Bournemouth, UK). Cell lines were maintained in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) with HEPES and L-glutamine (92.1, Mel202, PGA, CII, panc05.04, and NALM-6 cell lines) or IMDM (Lonza, Basel, Switzerland) with HEPES, L-glutamine (panc1), and supplemented with 10% fetal calf serum (FCS) and penicillin-streptomycin (Invitrogen) and incubated in 5% CO₂ at 37 °C. Panc05.04 was cultured in the presence of 1% Insulin-Transferrin-Selenium (ITS -G) (Thermo Fisher Scientific, Waltham, MA, USA). Primary CLL cells were thawed and cultured in IMDM (Lonza, Basel, Switzerland) with HEPES, L-glutamine, 10% FCS, and penicillin-streptomycin (Invitrogen) for functional experiments and incubated in 5% CO₂ at 37 °C.

Mouse spleens were collected and processed as described previously (14 (link)). CD8⁺ T cells were enriched from the splenocytes with a mouse CD8a⁺ T-Cell Isolation Kit (Miltenyi Biotec, Inc.). Then, the cells were expanded as described previously with modifications (22 (link)). Briefly, the cells were activated in 6-well plates (5 × 10⁶ cells/5 mL/well) coated with anti-CD3 (0.5 μg/mL, clone 2C11, Bio X Cell) and anti-CD28 (5 μg/mL, clone 37.51, Bio X Cell) for 20 hours in RPMI1640 medium containing 10% FBS and antibiotics and supplemented with 2 mmol/L L-glutamine, 50 μmol/L 2-mercaptoethanol, 1% insulin-transferrin-selenium (ITS-G, Thermo Fisher Scientific), 6 ng/mL mouse IL2 and 0.5 ng/mL mouse IL7 (ProSpec). After another 48 hours expansion, the cells were harvested and used for adoptive T-cell therapy in mice.