Figure 3 Expression of mutant STIM1 in cultured cells and its effect on store-operated Ca2+ influx Expression of mutant STIM1 in cultured cells and its.

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Figure 3 Expression of mutant STIM1 in cultured cells and its effect on store-operated Ca2+ influx Expression of mutant STIM1 in cultured cells and its effect on store-operated Ca2+ influx (A) C2C12 myoblasts were transfected with wild-type (WT) and the C-terminal inhibitory domain mutant STIM1 and labeled with anti-STIM1 antibody, phalloidin, and 4',6-diamidino-2-phenylindole (DAPI). WT STIM1 displays diffuse distribution in C2C12 cells, whereas the signal of the mutant STIM1 concentrates intensely around nuclei and exhibits an aggregation-like appearance in the shrunk cytoplasm. Bar = 50 μm. (B) Seven stitching images (5 × 5) of the C2C12 myoblasts were captured using a fluorescence microscope, and the number of cells with diffuse vs aggregation-like localization of STIM1 was counted. The majority of cells expressing WT STIM1 show diffuse localization, whereas most cells expressing the mutant STIM1 exhibit the aggregation-like pattern. (C) Intracellular Ca2+ concentration was measured in HEK293 cells expressing WT STIM1 (gray square; n = 40 cells), the mutant STIM1 (black triangle; n = 42 cells), or control (open circle; n = 47 cells). The perfusion solution was changed from HEPES-buffered saline (HBS) containing 2 mM Ca2+ to Ca2+-free solution containing 0.5 mM ethylene glycol tetraacetic acid (EGTA). To evaluate Ca2+ influx across the plasma membrane, 2 mM Ca2+ was applied to the cells. The duration of exposure to Ca2+-containing HBS and Ca2+-free HBS is indicated above the graph. (D) Maximal intracellular Ca2+ rises after readdition of 2 mM Ca2+ to the cells. Data points are mean ± SEM. p = 0.0028, 0.0104, and 0.5299 for WT vs mutant, control vs WT, and control vs mutant, respectively. Hidehiko Okuma et al. Neurol Genet 2016;2:e50 © 2016 American Academy of Neurology