Anti il 1β neutralizing antibody

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.

To evaluate the EMT-dependent induction of RFP expression in the interplay between CRC and inflammatory cells, HCT116-VRV3 and RKO-VRV3 cells (5 × 10⁴) were co-cultured with RAW264.7 mouse macrophages (5 × 10⁴). Twenty-four h later, lipopolysaccharide (LPS) (200 ng/ml) (Sigma-Aldrich) was added to the culture medium to induce the secretion of TNF-α and IL-1β by RAW264.7 cells. Anti–TNF-α neutralizing antibody (100 ng/ml) and anti–IL-1β neutralizing antibody (500 ng/ml) (Abcam) were added to the culture medium to attenuate the effects of TNF-α and IL-1β secreted by RAW264.7 cells. Then, 48 h later, the images of HCT116-VRV3 and RKO-VRV3 cells co-cultured with RAW264.7 cells were obtained in three randomly selected fields in each group using a confocal laser scanning microscope (FV10i; Olympus). Quantification of RFP expression was analyzed by calculating the integrated densities of RFP with ImageJ software.