FIGURE 7.

CN585 decreases NFAT reporter gene activity and reduces intracellular IL-2 production of stimulated PBMC. A, Jurkat cells were transfected with a NFAT-luciferase reporter plasmid by electroporation. The cells were preincubated with the compounds for 30 min and then stimulated with PMA/ionomycin for 5 h. After cell lysis the level of extracted luciferase was determined by using luciferase assay system. Additionally, pcDNA-lacZ was cotransfected, and β-galactosidase activity was measured as internal standard. B, Ficoll-isolated human PBMC (5 × 10⁶ cells/ml) were preincubated with various concentrations of CN585, 15 μm CN675, or 1 μm CsA at 37 °C for 30 min in a 24-well plate. Subsequently, cells were stimulated with PMA/ionomycin (iono) for 5 h and for the last 3 h in the presence brefeldin A. PBMC were fixed in paraformaldehyde, permeabilized with 0.5% saponin in PBS/fetal calf serum, incubated with anti-IL-2 fluorescein isothiocyanate-conjugated antibody, and analyzed by flow cytometry. The data presented are the means ± S.D. of three independent experiments.