(A) JPX-9 cells were untreated or stimulated with CdCl2 (15 μM) and cyclosporin A (1 μg/ml) or sc-514 (25 μm) for 24 hours. Real time PCR for CD40 was performed, and CD40 mRNA expression was normalized to 18S rRNA in the same real time PCR reaction. Error bars represent SEM, with n=3 for each condition. This experiment was repeated one additional time with similar results. (B) JPX-9 cells were treated as in Panel A, and real time PCR was performed for CD40L. CD40L mRNA expression was normalized to 18S rRNA in the same real time PCR reaction. (C) Jurkat-E6 cells were transfected with 0.25 μg NFAT luciferase reporter and 0.01 μg pRL-TK luciferase. Transfected cells were treated with or without PMA/ionomycin and cyclosporin A for 7 hours, harvested, and a dual luciferase assay was performed. NFAT luciferase values were normalized to renilla luciferase. Error bars represent SEM, with n=3 for each condition.