NCL-SG3 cells were stably transduced with shBEST2, shTMEM16A, or control shRNA (shCtrl). (A) TMEM16A and BEST2 mRNA expression measured by quantitative real-time PCR and normalized to levels in shCtrl-transduced cells. GAPDH was used as a housekeeping control. Mean ± SEM of 3 independent experiments done in triplicate. (B) [Ca²⁺]_i measurements in Fura-2–loaded NCL-SG3 cells stimulated with 1 μM ionomycin (Iono) in Ca²⁺-free Ringer solution followed by readdition of 1 mM Ca²⁺ to induce SOCE. Representative [Ca²⁺]_i traces (left) and AUC (right) after readdition of 1 mM Ca²⁺ (Ca²⁺ influx phase). For additional details see Supplemental Methods. Bar graphs represent mean ± SEM of 3 independent experiments done in triplicate. (C and D) [Cl^–]_i measurements in MQAE-loaded NCL-SG3 cells analyzed by single-cell time-lapse fluorescence microscopy. 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. The Ca²⁺ concentration in the extracellular buffer remained constant (1.8 mM) throughout the experiment. (C) [Cl^–]_i traces averaged from 50 cells in 1 experiment, and representative of 6–7 independent experiments. (D) Analysis of Cl^– efflux rates (420–600 seconds, 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 from data shown in C. For additional details see Figure 5E and Supplemental Methods. Data in D are mean ± SEM of 7 independent experiments for shCtrl, and 6 for both shBEST2 and shTMEM16A. Statistical analyses in A and D were performed using 1-way ANOVA followed by Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.