Supporting Information

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Fig. 7. Chemical structures of NFATc3 inhibitors. Additional data for these compounds are presented in Table 1.

Fig. 8. NFAT inhibitors block store-operated calcium (SOC)-dependent calcium influx. (A) Inhibition of ionomycin-induced sustained calcium influx in BHK cells. Fluo-3 labeled cells were pretreated with DMSO (control) or indicated compounds for 15 min in HBS with 2 mM calcium prior to addition of 1 μM ionomycin (arrow). EGTA (5 mM) was added 1 min prior to addition of ionomycin. Relative changes in intracellular calcium levels are represented as ratio F_t/F₀, where F_t and F₀ are the integrated fluorescence intensities at times t and zero. (B) Dose-dependent inhibition of thapsigargin-induced calcium influx in BHK cells. Fura-2 labeled cells are treated with varying concentrations of compound 4 for 1 and 3 min with thapsigargin in calcium-free HBS prior to addition of CaCl₂ (2 mM final) to evoke calcium influx. Fluorescence recordings 1 min before readdition of CaCl₂ are shown. Each graph is a representative trace of several independent experiments.

Fig. 9. Prior store depletion does not affect the inhibitory effect of compounds on SOC-dependent calcium influx. Fluo-4 labeled Do11 cells were stimulated for 4 min with thapsigargin and then with indicated compounds (arrow) in calcium-free HBS prior to readdition of calcium (2 mM final) to allow calcium entry through SOC channels.

Fig. 10. Inhibition of calcium mobilization by NFAT inhibitors is independent of K channels and mitochondrial calcium homeostasis. (A) Do11 cells were stimulated as in Fig. 4a, except that high K buffer (145 mM KCl was substituted for NaCl) was used. (B) Do11 cells were stimulated as in Fig. 4a, except that CCCP was present throughout as indicated.

Fig. 11. Effect of NFAT inhibitors on SOC-dependent calcium mobilization. (A) Fluo-4 labeled Do11 cells were treated with DMSO (control) or indicated compounds for (arrow) 1 min prior to addition of 1 μM thapsigargin (arrow) in calcium-free HBS. After 1 min, calcium mobilization through SOC was induced by addition of calcium (2 mM final). Relative changes in intracellular calcium levels are represented as ratio F_t/F₀, where F_t and F₀ are the integrated fluorescence intensities at times t and zero. (B) Same as A except that compounds were treated 3 min before thapsigargin addition.