Fig. 3. Requirement of NFAT3 for COX-2 but not iNOS induction, by TNF- in Cl41 cells. (A,B) 2x10⁵ cells of Cl41 transfectants were seeded into each well of a 6-well plate, and the cells were extracted with Tris-Glycine SDS sample buffer after the cell density reached 90-100%. Western blotting was carried out with specific antibodies as indicated. NF-B and c-Jun were used as specific controls for NFAT3 siRNA, and GAPDH was used as the protein loading control. (C-F) Cl41-COX-2 mass1, Cl41-siNFAT3/COX-2 mass1, Cl41-iNOS mass1 cells and Cl41-siNFAT3/iNOS mass2 (8x10³), were seeded into each well of a 96-well plate. The cells were treated with TNF- for 72 hours and the luciferase activities were determined. The results were expressed as COX-2 (C,E) and iNOS (D,F) transcriptional induction relative to medium control (relative COX-2 induction or relative iNOS induction). Each bar indicates the mean and standard deviation of triplicate wells. (G) Cl41 COX-2 mass1 and Cl41 siNFAT3/COX-2 mass1 (2x10⁵) were seeded into each well of a 6-well plate, and cultured in 5% FBS MEM at 37°C. When the cell density reached 70-80%, the cells were exposed to various concentrations of TNF- as indicated for 24 hours and 48 hours. The induction of COX-2 at protein level was detected by western blot.