Figure 2: Orai2-deficient cells have increased SOCE.

(a,b) Analysis of Ca²⁺ store depletion and SOCE following thapsigargin (TG) stimulation in naive T cells from WT, Orai1-deficient (Orai1^fl/flCd4cre), Orai2-deficient (Orai2^−/−) and Orai1/Orai2-deficient (Orai1^fl/flOrai2^−/−Cd4cre) mice using a FlexStation plate reader. (b) Quantification of store depletion (area under the curve, AUC_{120 s–400 s}) and SOCE (peak F340/380) as shown in a; means±s.e.m. of seven to nine mice. (c) Indirect measurement of SOCE by Mn²⁺ quenching of Fura-2 fluorescence. WT and Orai2-deficient naive T cells were stimulated with TG in Ca²⁺-free Ringer solution followed by the addition of 1 mM Mn²⁺-containing Ringer solution. Quantification of the slope of Mn²⁺ quenching in TG-stimulated T cells normalized to quenching in non-stimulated cells; means±s.e.m. of three mice. (d–g) Increased SOCE in human fibroblasts after shRNA-mediated knockdown of ORAI2 is not due to ORAI1 upregulation. (d) Analysis of knockdown efficiency of ORAI1 and ORAI2 in human fibroblast cells after transduction with shRNAs against ORAI1 and ORAI2 by qRT–PCR; means±s.e.m. of four experiments. (e) Analysis of Ca²⁺ store depletion and SOCE following TG stimulation in shORAI1- or shORAI2-treated human fibroblast cells using a FlexStation plate reader. (f) Quantification of store depletion (AUC_{120 s–600 s}) and SOCE (F340/380 peak and F340/380 slope_{600 s–640 s}) in human fibroblast cells as shown in e; means±s.e.m. of four experiments. (g) Analysis of ORAI1 surface expression in human fibroblast cells following shRNA-mediated knockdown of ORAI1 and ORAI2 as described in (d) by flow cytometry using an anti-ORAI1 monoclonal antibody (29A2) recognizing the second extracellular domain of ORAI1; means±s.e.m. of three experiments. *P<0.05; **P<0.005; ***P<0.001 in (b–d,f,g) using unpaired Student’s t-tests.