(A) Analysis of Stim1 and Stim2 gene expression in enamel organ (EO) cells of WT and Stim1/2^K14cre mice by qRT-PCR showing significantly decreased levels of both transcripts in the latter. Data represent mean ( ± SEM) of n = 4 WT and n = 6 Stim1/2^K14cre mice (***P < 0.001, 2-tailed unpaired Student’s t test). (B) STIM1 protein expression in maturation-stage ameloblasts, endothelial cells, salivary glands, muscle, and brain of WT and Stim1/2^K14cre mice by immunofluorescence. Strong positive reactions were detected in all WT tissues, whereas STIM1 signals were found only in nonkeratin 14 targets (i.e., muscle and brain). Scale bar: 20 μm. (C–F) Conditional deletion of Stim1 and Stim2 genes drastically diminishes store-operated Ca²⁺ entry (SOCE) in ameloblast cells. EO cells isolated from WT and Stim1/2^K14cre mice were loaded with Fura-2-AM and intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were determined by time-lapse microscopy after thapsigargin (TG) stimulation and readdition of 2 mM Ca²⁺. (C) Representative images of EO cells from WT and Stim1/2^K14cre mice showing the F340/380 fluorescence ratio before stimulation with TG in Ca²⁺-free medium (5 seconds) (baseline) and after addition of 2 mM Ca²⁺ (490 seconds) (peak SOCE). Scale bar: 20 μm. (D–F) Upon stimulation with TG (1.25 μM), Stim1/2^K14cre EO cells (red tracings) showed significant differences in ER Ca²⁺ release and Ca²⁺ entry relative to controls (black tracings). Data were normalized by setting baseline to 1. Statistical analysis of average delta peak for TG-induced store depletion and for Ca²⁺ entry are shown in E and F, respectively. Data in D–F represent mean ± SEM from n = 5 different experiments. Each experiment sampled cells obtained from 1 WT mouse and 1 Stim1/2^K14cre littermate. Total cells: WT = 132, Stim1/2^K14cre = 126. (**P < 0.005, ***P < 0.001, 2-tailed unpaired Student’s t test).