Figure 8. Efficient inhibition of NFAT1 activation by CsA and FK506. (A) CACO-2 cells were treated with inhibitors at concentrations depicted for 30 min at 37°C. Cells were then kept unstimulated as control (CT) or stimulated with 2 μM of Iono for 3 min, lysed and the total protein extract was analyzed by SDS-PAGE. Western blot for NFAT1 followed. (B) CACO-2 cells were transfected with pGL4.30-NFAT-Luc and pRL-TK, and treated with CsA (2 μM), FK506 (2 μM) or Rapa (20 nM) for 24 h. Cells were stimulated for the last 6 h of culture with PMA (20 nM) and Iono (2 μM), lysed and luciferase activity was measured. Results are the mean of four independent experiments. * marks p < 0.05 when comparing to unstimulated group. (C) CACO-2 cells were transfected with pGL4.30-NFAT-Luc and pRL-TK, with or without pEGFP-VIVIT. Starting at 24 h after transfection (0 h), cells were stimulated for the last 6 h of culture with PMA (20 nM) and Iono (2 μM), lysed and luciferase activity was measured. Data represents the average of two independent experiments. (D) CACO-2 cells were transfected with pEGFP-VIVIT or mock-treated. Twenty-four hours after transfection cells were replated, and growth was analyzed by staining with violet crystal. Results are the mean of five independent experiments.