Fig. 1.

CD95-mediated Ca²⁺ response prevents DISC formation. (A) Ca²⁺ response to CD95L (100 ng/mL) measured ratiometrically in suspensions of Indo-1–loaded activated PBLs. Cells were preincubated with BAPTA-AM (5 μM, Upper) or 2-APB (44 μm, Lower), or left untreated (control) before the addition of CD95L, indicated by the black arrow. The data represent mean ± SD of four independent experiments. (B) Jurkat, H9, and activated PBLs were pretreated as in A with BAPTA-AM, 2-APB, or DMSO (control), and then stimulated (15) for 15 min with the agonist anti-CD95 mAb APO1-3 (1 μg/mL) or left untreated (0). CD95 was immunoprecipitated, the immune complex was resolved by SDS/PAGE, and the indicated immunoblotting was performed. Total lysates were loaded as a control. p41/43 corresponds to the first step of caspase-8 cleavage. (C) Ca²⁺ signal and DISC formation in ionomycin-treated T cells. (Upper) Indo-1 ratiometric fluorescence in Jurkat T-cell suspensions exposed to ionomycin (1 μM) before stimulation with CD95L (100 ng/mL) at the indicated times. Data are mean ± SD of four independent experiments. (Lower) CD95 immunoprecipitation in Jurkat cells treated as in C, Upper. Data are representative of three independent experiments. (D) [Ca²⁺]_i in cell populations recorded with Indo-1 in activated PBLs stimulated with CD95L (100 ng/mL) in control medium (0.8 mM Ca²⁺; black trace) or in medium containing BAPTA to chelate free Ca²⁺ (0.8 mM Ca²⁺ supplemented with 2 mM BAPTA; gray trace). Data represent mean ± SD of three independent experiments. The area under the curve corresponds to 321 ± 40 in regular medium 148 ± 37 in Ca²⁺-free medium (P < 0.001). (E) DISC formation in activated PBLs preincubated for 30 min in medium containing 0.8 mM Ca²⁺ or medium supplemented with 2 mM BAPTA, then stimulated with APO1-3 (1 μg/mL). The star corresponds to the heavy chain of the APO1-3 IgG3.