FIGURE 3.

The role of NF-AT for IL-22 expression as detected in T/A-stimulated Jurkat T cells. A, Jurkat T cells were transfected for 5 h with the indicated pGL3-IL22 promoter constructs (Prom1–4) together with Renilla luciferase as described under “Experimental Procedures.” After 15 h of rest, cells were either kept as unstimulated control or stimulated with T (100 ng/ml)/A (10 μm). After another 8 h, cells were harvested and luciferase assays were performed. Means of luciferase activity are shown as fold-induction compared with unstimulated control (transfected with the same plasmid) ± S.D. (n = 5); **, p < 0.01 compared with unstimulated control (transfected with the same plasmid); ##, p < 0.01 compared with pGL3-Prom3 upon T/A stimulation; raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction. B, Jurkat T cells were transfected for 5 h with pGL3-Prom3 together with Renilla luciferase as described under “Experimental Procedures.” After 15 h of rest, cells were either kept as unstimulated control or stimulated with T (100 ng/ml)/A (10 μm) for another 8 h. Where specified, cells were preincubated for 1 h with the indicated concentrations of CsA before T/A addition. Thereafter, cells were harvested and luciferase assays were performed. Means of luciferase activity are shown as fold-induction compared with unstimulated control ± S.D. (n = 4); **, p < 0.01 compared with unstimulated control; ##, p < 0.01 compared with T/A alone; raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction. C, Jurkat T cells were transfected for 5 h with pGL3-Prom3 or pGL3-Prom3 displaying mutated NF-ATS1–4 (PM) together with Renilla luciferase as described under “Experimental Procedures.” After 15 h of rest, cells were either kept as unstimulated control or stimulated with T (100 ng/ml)/A (10 μm) for 8 h. Thereafter, cells were harvested and luciferase assays were performed. Means of luciferase activity are shown as fold-induction compared with unstimulated control (transfected with the same plasmid) ± S.D. (n = 3); **, p < 0.01 compared with unstimulated control (transfected with the same plasmid); #, p < 0.05 compared with unmutated pGL3-Prom3 stimulated with T/A; raw data were analyzed by one-way ANOVA with post hoc Bonferroni correction. D, Jurkat T cells were either kept as unstimulated control or stimulated with T (100 ng/ml)/A (10 μm). After 4 h, cells were harvested followed by ChIP analysis for detection of NF-ATc1, NF-ATc2, and c-Rel binding to NF-ATS4 as described under “Experimental Procedures.” One representative of three independently performed experiments is shown. All cultures were adjusted to a final concentration of 0.1% DMSO (vehicle for T/A, CsA).