Figure 3.

Mitochondria are necessary for sustained Ca²⁺ signaling and NFAT translocation. [Ca²⁺]_i and EGFP fluorescence were measured in parallel in fura-2-loaded Jurkat cells expressing NFATc1-EGFP. (A) Images of NFATc1-EGFP fluorescence are shown for cells before (Left) or 10 min after stimulation with OKT3 (Right; 1:75 ascites). In unstimulated cells, NFATc1-EGFP is mostly cytosolic, and OKT3 elicits extensive translocation of NFATc1-EGFP to the nucleus. (B, C) [Ca²⁺]_i and EGFP fluorescence measured in the absence (B) or presence (C) of 1 μM CCCP in single cells at room temperature. After depletion of Ca²⁺ stores for 10 min in 0-Ca²⁺ + 1 μM TG, 2 mM Ca²⁺ was readded as indicated. (D) [Ca²⁺]_i and nuclear NFATc1-EGFP intensity in OKT3-stimulated cells at 37°C. A maximally effective concentration of OKT3 (ascites diluted 1:75) was added to the cells as indicated under control conditions (solid trace) or in the presence of 1 μM CCCP (dotted trace). Each trace is an average of three experiments performed on a single day comprising a total of 151 (control) and 146 (+CCCP) cells.