Fig. 3

STIM1 localizes near phagosomes and promotes global and local Ca²⁺ signals in BMDCs. a, b Stim1 ablation strongly decreased SOCE in immature and CpG-matured BMDCs, measured as the slope of Ca²⁺-re-entry after store depletion with 1 μM Tg in Ca²⁺-free medium and 2 mM Ca²⁺ re-addition a, or as the area under the curve (AUC) of the first 3 min after acute exposure to the agonist PAF (2 μM, b). Traces are averages of 11–23 cells. N = 10/6; 3/4 (coverslips each) containing 129/119; 26/70 (WT) or 139/98; 67/97 (Stim1 ^−/−) cells for Tg: CTR/CpG; PAF: CTR/CpG. c, d Localized Ca²⁺ signalling near phagosomes (green dots, arrows, d) was measured in BMDCs loaded with 4 μM Fluo-8 and 2.5 μM BAPTA after 30 min or 90 min of exposure to targets, and in the absence or presence of 10 μM GSK or 1 μM Xesto after 30 min (c). Stim1 ablation reduced periphagosomal Ca²⁺ hotspots at 30 but not 90 min, and both GSK and Xesto reduced hotspot frequency in WT but not Stim1 ^−/− cells (c). N = 5/4;7/4;5/3;6/4 coverslips representing 870/794; 1916/1154; 898/814; 1124/1372 phagosomes; 196/178; 370/238; 270/265; 375/211 cells for WT/Stim1 ^−/− 30;90:30+GSK;30+Xesto. The colour-coded ratio images are Fluo-8 fluorescence/average cytosolic Fluo-8 fluorescence and show representative hotspots (d). e BMDCs transduced with mCherry-STIM1 (magenta) and loaded with Fluo-8 (green) as above display periphagosomal accumulations of STIM1 fluorescence that co-localize with Ca²⁺ hotspots (white arrows), as well as puncta that show no Ca²⁺ activity (yellow arrows). 20:1 target:cell ratio. Bars = 3 μm. Error bars are means + SEM, *p < 0.5, **p < 0.1 using a Student’s t-test