FIGURE 3.

Curcumin inhibits NFAT transcriptional activity by blocking its nuclear expression. A, curcumin inhibits NFAT-mediated transcription. Jurkat T cells were transfected with luciferase constructs containing multiple copies of the NFAT binding element derived from the IL-2 promoter or the consensus DNA binding element of either AP1 or NF-κB. After overnight recovery, the cells were split and pre-incubated with the indicated amounts of curcumin for 0.5 h and then stimulated with PMA (5 ng/ml) and ionomycin (0.5 μm) or left unstimulated. Luciferase activity was determined after 8 h stimulation. Data are representative of three independent experiments performed in triplicate. B, curcumin prevents stimulation-induced nuclear expression of NFAT determined by Western blot. Jurkat T cells were stimulated with PMA and ionomycin for 2 h in the absence or presence of different concentrations of curcumin as indicated. Nuclear proteins were isolated and the expression levels of NFAT were analyzed by Western blot with the specific mAb against NFATc1. YY1 was used for an equal loading control. Total cell lysates were used as an additional control for total amounts of NFAT expression in the experiment. C and D, as controls, Western blot was also carried out in parallel with antibodies against Jun and NF-κB (p50/p65), respectively. Total cell lysates were used as additional controls for total amounts of Jun and NF-κB expression in the experiment.