Fig. 1: SOCE but not ORAI1 is required for maintenance of Ca²⁺ oscillations.

a–e Representative Ca²⁺ oscillations in response to 10 µM carbachol (Cch) measured using Fura2 in wild-type HEK293 and ORAI-TKO cells. Cells were maintained in HBSS containing 2 mM Ca²⁺ and stimulated with 10 µM Cch at 1 min (indicated by arrow in “a”). Representative traces from five cells/condition were chosen to represent the datasets as a whole. In c, d, cells were treated with 5 µM Gd³⁺ to block SOCE. In e, ORAI-TKO cells were treated with 1 mM Gd³⁺ (so-called Gd³⁺ insulation to block both SOCE and Ca²⁺ extrusion). f Quantification of total oscillations/14 min for all conditions from a–e (from left to right n = 80, 84, 180, 127, and 78; n-values correspond to individual cells). g–l Representative Ca²⁺ oscillations in response to 10 µM Cch measured using Fura2 over the course of 15 min in ORAI single (SKO) and double (DKO) knockout cell clones. m–p Quantification of total oscillations/14 min (m) (from left to right n = 190, 111, 58, 29, 206, 214, and 67; n-values correspond to individual cells), % of oscillating cells (n), % of plateau cells (o), and % of non-responding cells (p) for data in g–l (for n–p, from left to right n = 12, 6, 6, 6, 9, 6, and 9; n-values correspond to independent experiments). q Direct ER Ca²⁺ measurements using CEPIA1er in parental HEK293 cells and ORAI-DKO cells. r–w Measurements of ER Ca²⁺ release (in 0 mM Ca²⁺) and SOCE (in 2 mM Ca²⁺) on store depletion with 2 µM thapsigargin (Tg) in parental HEK293 cells and ORAI-SKO and DKO cells; all traces are plotted as mean ± SEM. x Quantification of SOCE magnitude for all conditions from r–w. Scatter plots (f, m, x) are represented as mean ± SEM and were statistically analyzed using a Kruskal–Wallis one-way ANOVA with multiple comparisons to WT HEK293 where (*p < 0.05, ****p < 0.0001) (from left to right n = 413, 244, 243, 217, 256, 179, and 435; n-values correspond to individual cells). Where indicated, an independent Kruskal–Wallis one-way test was performed comparing ORAI2-3-DKO cells to ORAI2-SKO and ORAI3-SKO cell lines. Scatter plots (n–p) are represented as mean ± SEM and were statistically analyzed using an ordinary one-way ANOVA with multiple comparisons to WT HEK293 (*p < 0.05).