Fig. 2

STIM1 promotes Ca²⁺-dependent migration in vivo and in vitro. a CD45.1 congenic wild-type hosts were injected with a mixture of 1 × 10⁶ wild-type GFP+ (WT-GFP), 1 × 10⁶ Stim1 ^−/− BMDCs and 0.5% OVAb. DL were isolated and the relative percentage of WT:Stim1 ^−/− (CD11c⁺, CD45.2⁺) cells analysed by flow cytometry. Full gating strategy is shown in Supplementary Fig. 2a. The proportion of Stim1 ^−/− cells was significantly reduced 24 h but not 48 h after injection. N = 4 pairs of mice. b A 24 h transwell migration assay revealed 40–65% increases in migration towards fMIFL, SDF-1 and CCL21 chemoattractants in WT cells. Stim1 ^−/− BMDC showed reduced migration towards fMIFL and SDF-1 but not CCL21 (all applied at 10⁻⁶M) as compared to WT. Full gating strategy is shown in Supplementary Fig. 2b. N = 3 in triplicate wells. c Ca²⁺ transients induced by acute exposure (arrows) of chemoattractants, as measured using Fura-2 (traces representative average of 14–22 cells). Quantification of the area under the curve (AUC) of the first 2 min after chemoattractant addition (right bottom panel) revealed significantly lower Ca²⁺ entry in Stim1 ^−/− BMDCs in response to fMIFL and SDF-1, while no Ca²⁺ entry in response to CCL21 was detected for either genotype. N = 4 (coverslips each) containing: 68/66/72 (WT) or 47/56/52 (Stim1 ^−/−) cells for fMIFL/SDF-1/CCL21. Error bars are means + SEM, *p < 0.5, **p < 0.1 using a two-way ANOVA and Sidak’s post test for a and a Student’s t-test for b and c