Figure 2

Increased Ca²⁺ levels and Ca²⁺-dependent signaling in Nptn ^−/− T cells. (A) Flow cytometric ratiometric Ca²⁺ measurements in T cells. Anti-CD3 labeled CD4 T cells in buffer containing 1 mM Ca² were stimulated by crosslinking CD3 (anti-hamster). The diagram shows kinetics of the mean instantaneous single cell ratio ± SD of wt and Nptn ^−/− naive CD4 T cells from 3 experiments. The bar graph shows the mean normalized baseline ratios ± combined SD from 5 experiments, ***p < 0.001, unpaired two-tailed t-test. Further quantifications of baseline and peak levels and the decay phase are summarized in Supplementary Table S1. (B) Confocal imaging of nuclear NFAT localization. Images show NFAT immunofluorescence and DAPI staining in wt and Nptn ^−/− T cells at different magnifications from separate samples. Scale bars: left, 50 μm; right, 20 μm. Normalized nuclear NFAT intensities of 1299 wt and 1456 Nptn ^−/− single cells from 5 experiments are presented, overlay bars showing mean normalized values ± combined SD from these 5 experiments, ***p < 0.001, unpaired two-tailed t-test. (C) Detection of cytokines in stimulated wt and Nptn ^−/− T cells by intracellular FACS. Representative FACS plots show IL2 and IFNγ expression in CD4 T cells cultured for 3 or 6 days on immobilized anti-CD3. Mean proportions of IL2 and IFNγ producing cells ± SD derived from 3 and 8 experiments, respectively, are shown. *p < 0.05, ***p < 0.001, unpaired two-tailed t-test.