NFATc3 up-regulates the transcriptional activity of IL2 and COX2 promoters. (A-B) Jurkat cells were infected with the indicated knock-down LV vectors, and 48 hours later were transiently transfected with a luciferase reporter construct containing the IL2 (A) or the COX2 (B) promoter. After 16 hours, cells were stimulated for 5 hours with PMA + Io and luciferase activity was measured. Luciferase activity is shown in relative light units. Data are means ± SD (n ≥ 3); **P < .01 and ***P < .001 compared with control (KDSc). (C) Jurkat cells expressing KDSc or KD-c3 were transfected with the COX2-luc reporter construct plus an expression plasmid encoding wild-type NFATc1 (c1), wild-type NFATc3 (c3), or knock-down–resistant NFATc3 (c3Res). After 16 hours, cells were stimulated with PMA + Io for 5 hours and luciferase activity was measured. Luciferase activity is shown as the -fold induction relative to nonstimulated cells. One representative experiment of 4 is shown. (D-E) ChIP assays of NFATc1 and NFATc3 in Jurkat cells stimulated for 30 minutes with PMA + Io; histograms show the increased binding of both transcription factors to the endogenous promoters of IL2 (D) and COX2 (E). Data represent the amount of chromatin precipitated with αNFATc1 or αNFATc3 antibody compared with the input and are normalized to values obtained with IgG control antibodies. The results shown are representative of at least 3 experiments.