Fig. 2: ORAI3 and ORAI2 are required for Ca²⁺ oscillations.

a–h Representative Ca²⁺ oscillations in response to increasing concentrations of carbachol (1–30 µM Cch) measured using Fura2 in parental HEK293 cells, and in single, double, and triple ORAI knockout cells. Cells were maintained in HBSS containing 2 mM Ca²⁺ for the duration of the experiments and stimulated with 1, 3, 10, and 30 µM Cch for a duration of 5 min each. Representative traces were chosen from five cells/condition to represent the datasets as a whole. i–n Quantification of % of oscillating cells (i, l), % of plateau cells (j, m), and % of non-responding cells (k, n) for conditions from a–h (for i–k and l–n, n = 6, 3, 3, and 3 from left to right; each point represents an independent experiment). Quantification of oscillation data was performed using one-way ANOVA with multiple comparisons to WT HEK293 cells (*p < 0.05). o Representative Western blot documenting ORAI1 protein knockdown with siRNA in WT HEK293 cells. p Quantification of ORAI1 protein knockdown using ORAI1/GAPDH densitometry. Each data point represents a technical replicate from four independent knockdown experiments (n = 12). q Representative Ca²⁺ oscillations in response to 10 µM carbachol (Cch) followed by measurements of SOCE elicited by subsequent addition of 2 µM thapsigargin in HEK293 cells transfected with siRNA against ORAI1 (siORAI1) and non-targeting control siRNA (siNT). r Quantification of the magnitude of SOCE in siNT- and siORAI1-transfected cells (n = 240 for siNT and 256 for siORAI1; each point represents an individual cell). s–v Quantification of total oscillations/14 min (s), % of oscillating cells(n = 144 for siNT and 182 for siORAI1; each point represents an individual cell) (t), % of plateau cells (u), and % of non-responding cells (v) in siNT- and siORAI1-transfected cells (for t–v, n = 6 for both siNT and siORAI1; each point represents an independent experiment). All siRNA scatter plots, which represent the mean ± SEM were analyzed using the Mann–Whitney U-test (*p < 0.05; ***p < 0.001; ****p < 0.0001; ns, not significant).