Figure 3.

Cytosolic and subplasmalemmal Ca²⁺ levels in response to KCl stimulation were perturbed in MICU2-silenced INS-1 832/13 cells. Cytosolic Ca²⁺ levels ([Ca²⁺]c) in MICU2-silenced INS-1 832/13 cells (and scramble control) upon stimulation with 36 mM of KCl as measured by Fluo4 (A and B). Average traces are shown in A, while B shows differences in maximal Fluo4 signals. (C) Image of mem-Case12 staining, n. b. specific membrane localization. Submembrane Ca²⁺ levels of [Ca²⁺]_mem in MICU2-silenced INS-1 832/13 cells upon stimulation with 36 mM of KCl measured using mem-case12 (D and E); average traces are shown in D, while E shows iAUC quantification of KCl's effect on [Ca²⁺]_mem. In (F and G), similar experiments are shown but using oligomycin and antimycin to block mitochondrial Ca²⁺ uptake prior to KCl addition, with oligomycin applied alone as a control. Mean ± SEM are given. (H) Example of line scan selection: the white arrow through the center of the cell is the only part of the field of view that was imaged; the accompanying trace within the white rectangle shows the fluorescence signal for each corresponding point along the arrow. (I) Mean Ca²⁺ intensity across INS-1 832/13 cells ∼500 ms after initiation of response to 36 mM of KCl. Cells had different sizes, so the data were interpolated to standardize each cell to a set number of points, allowing for comparison. The X axis represents the cell axis moving from one side of the plasma membrane (−50) through the center of the cell (0) to the opposite side of the plasma membrane (50). Points close to the extremities represent the submembrane region. (J) Average trace of cytosolic Ca²⁺ at cell edge and cell center in MICU2-silenced INS-1 832/13 cells (and scramble control). (K) iAUC quantification of [Ca²⁺]_c at the cell edge and cell center in MICU2-silenced INS-1 832/13 cells (and scramble control). Subplasmalemmal Ca²⁺ levels ([Ca²⁺]_mem) in MICU2-silenced INS-1 832/13 cells (and scramble control) upon stimulation with 36 mM of KCl. (L) Insulin secretion in control and Micu2-silenced INS-1 832/13 cells after 15 min of incubation in 36 mM of KCl. Comparisons were made with an unpaired two-tailed Student's t-test. In 3L, statistical comparisons were made by a paired two-tailed Student's t-test since variability in short-term incubations was greater. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.