FIGURE 2.

NFAT1 activation and NFAT-dependent regulation of gene expression are increased by switching phSG cells to KGM-H medium. A and B, AdCMVGFP-NFAT1 (7.5 × 10⁶ MOI) was used to mediate expression of NFAT1 in phSG cells. Cells were infected with the vector and maintained in either high (A) or low (B) calcium medium for 48 h. Cells were treated with Tg (1 μm), and GFP-NFAT fluorescence was measured in the cytosol (insets) and nucleus for the indicated time period. Values are shown relative to the initial fluorescence in these two cellular areas. C, the average intensity of the GFP signal in the nuclei in cells in KGM-H and KGM-L recorded at 30 min was calculated as (F₃₀ − F₀)/F₀. The number of cells examined was as indicated. D, Tg-stimulated [Ca²⁺]_i increases in cells maintained in KGM-L or KGM-H. E, bar graph showing the amplitude of sustained [Ca²⁺]_i at t = 400 s in both sets of cells. Amplitude was calculated as (F₄₀₀ − F₀) in arbitrary fluorescence units. F, NFAT-RE-Luc (pGL-4.30, white) or NFκB-RE-Luc (pGL-4.32, black) was co-transfected with TK-Ren into phSG cells for 16 h before the medium was replaced with either KGM-L or KGM-H. Cells were then further incubated for another 6 or 8 h prior to luciferase measurements (see “Experimental Procedures” for details of the assay). *, p < 0.05. G, effect of knocking down endogenous NFAT on NFAT-Luc activity. Cells were co-transfected with the NFAT-Luc plasmid and either the scrambled siRNA (siN.C., white) or siNFAT1 siRNA (black) in KGM-L or KGM-H medium for 48 h. Data are shown relative to activity in control (cells transfected with siN.C. in KGM-L). Data are mean ± S.E. from triplicate samples of three separate experiments. *, p < 0.05. H, Tg stimulation of NFAT-dependent gene expression in phSG cells in KGM-H. Cells expressing NFAT-Luc were treated with (black) or without (white) Tg (1 μm) for 30 min, washed, and maintained in KGM-H for 16 h before being used in the luciferase assay. *, p < 0.05.