Figure 5. SOCE mediates Cl^– secretion in human sweat gland cells.

(A–C) NCL-SG3 cells were stably transduced with shORAI1, shSTIM1, control shRNA or left untransduced. (A) ORAI1 (left) and STIM1 (right) mRNA expression measured by quantitative real-time PCR. mRNA expression in shORAI1- and shSTIM1-transduced cells was normalized to that in nontransduced cells (no shRNA). GAPDH mRNA was used as a housekeeping control. Shown are the mean ± SEM of 5 independent experiments done in triplicates. (B) Representative [Ca²⁺]_i measurements in NCL-SG3 cells. Fura-2-loaded cells were stimulated with 1 µM ionomicyn (Iono) in Ca²⁺-free Ringer solution followed by readdition of 1 mM Ca²⁺ to induce SOCE. For additional details see Supplemental Methods. (C) Peak of [Ca²⁺]_i after store depletion (left) and SOCE (right) in NCL-SG3 cells. Data are the mean ± SEM of 6 independent experiments done in duplicates. (D and E) [Cl^–]_i measurements in MQAE-loaded NCL-SG3 cells. Cells were stimulated with 1 µM ionomycin (Iono) to induce SOCE in buffer containing 100 mM Cl^– followed by removal of extracellular Cl^– to force Cl^– secretion from the cells. (D) Averaged [Cl^–]_i traces from 50 cells in 1 experiment, and representative of 5 independent experiments. (E) Analysis of Cl^– efflux rates (left), reduction in [Cl^–]_i (middle; calculated as the integrated area_420-720s relative to baseline) and net Cl^– efflux (right) after removal of extracellular Cl^– at 420 seconds as shown in D. Net Cl^– efflux was calculated as mean baseline [Cl^–]_i (0–90 seconds) minus mean [Cl^–]_i (500–700 seconds). Data shown are the mean ± SEM of 5 independent experiments. (F) mRNA expression of TMEM16A, BEST2, NKCC1, AQP5, FOXA1 and NFATc3 in shRNA-transduced NCL-SG3 cells analyzed by quantitative real-time PCR. Data shown are the mean ± SEM of 4-5 independent experiments done in triplicates. Statistical analyses in A, C, and E were performed using 1-way ANOVA followed by Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.