Modulation of Chondrocyte Inflammation

Human chondrocytes were seeded in a 24-well plate at a density of 1 × 10⁵cells/well with DMEM and 10%FBS. The next day, transfection of MALT1 plasmids or siRNA was done using Lipofectamine. The chondrocytes were transfected with scrambled RNA (scRNA), LPCAT3 siRNA(200 nM),or MALT1 siRNA (200 nM) using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher, MA, USA) in 500μl RPMI medium for 8 h as described in our previous studies [22 (link), 23 (link), 65 (link)]. Then the medium was replaced with serum-richDMEM medium and cultured for 48h or as described in the figure legends. Similarly, 1 μg of pCMV6-XL4-MALT1 or pCMV-XL4 empty plasmids (Origene, MD, USA) were transfected using Lipofectamine 2000 (Thermo Fisher, MA, USA) in DMEM medium without antibiotics for 12h. Later the medium was replaced with fresh medium. 48 h post-transfection, the cells were stimulated with IL-1β as described in figure legends, and cell medium was collected for analysis of cytokines and eicosanoids. Alternatively, the cells were lysed in the SDS-PAGE sample buffer, and an equal amount of protein or volume was subjected to SDS-PAGE, followed by immunoblot analysis. The immunoblots were developed with chemiluminescence using HRP-substrate (Sigma). Densitometric analysis was performed using the Bio-Rad Chemi XRS system and Image J software.