Figure 2

STIM1/2 calcium sensors are required for muscarinic-induced SOCE and [Ca²⁺]_i response in human OPCs. hOPCs were transfected with STIM1 and/or STIM2, or scrambled siRNA, cultured in mitogen-containing media, and RNA was extracted after 24–48 h. For calcium imaging experiments, hOPCs were initially infected with GCaMP6s lentivirus and then transfected with STIM RNAi or scrambled control siRNA prior to time-lapse microscopy. RT-qPCR analysis of STIM1 (A) or STIM2 (B) mRNA expression relative to scrambled control transfected hOPCs following STIM1, STIM2 or STIM1/2 RNAi knockdown (STIM KD) (mean ± SEM, n = 7–8 human samples). Control normalized mean ± SEM, one-sample t test, *p < 0.05, **p < 0.01, ****p < 0.0001. (C) Representative pseudo-colored fluorescence time-lapse images of peak Oxo-M-induced calcium responses in siRNA transfected hOPCs cultured in calcium-free conditions. Images represent peak Oxo-M [25 µM] induced ER-store depletion (red text and arrows in (C) and (D) respectively) and SOCE following calcium re-addition (green text and arrows in (C) and (D) respectively). (D) Calcium traces of Oxo-M induced calcium response following siRNA transfection showing normalized mean pixel intensity ± SEM (blue shading) of n ≥ 30 cells per condition from one representative biological replicate. Timings for addition of Oxo-M and Ca²⁺-containing solution are indicated by horizontal bars above plots. (E) SOCE was measured as the total area under the curve following calcium re-addition normalized to ER-release of control cells in the matched biological replicate. STIM1/2 KD and pharmacological SOCE inhibition both resulted in a significant reduction in muscarinic-induced SOCE relative to control hOPCs. Cells were analyzed across 1–2 imaging fields in a single well per each condition and all responses were averaged per condition for each biological replicate. Data are presented as mean ± SEM of averaged cell responses per each of four independent experiments, with > 140 total cells quantified per each condition, n = 4 independent human fetal sample preparations. RM one-way ANOVA with Holm–Sidak’s post-hoc test, *p < 0.05, **p < 0.01). (F,G) The net effect of pharmacological SOCE inhibition or STIM KD on Oxo-M-induced increased [Ca²⁺]_i was determined in calcium-containing normal growth media by time-lapse microscopy. STIM1 KD or STIM2 KD hOPCs displayed a reduction in Oxo-M-induced calcium responses as denoted by reduced average AUC (F) as well as a reduction in oscillatory frequency relative to control hOPCs (G). Cells were analyzed across two imaging fields in a single well per each condition and all responses were averaged per condition for each biological replicate. Data represent log₁₀-transformed mean ± SEM of per cell responses from four independent experiments (n = 4 independent human fetal sample preparations), with > 100 total cells quantified per each condition. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, linear model with Tukey’s HSD posttest. Scale: 50 µm.