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Fig. 7. ML-9 reverses constitutive EYFP-D76N/D78N-Stim1 localization and SOCE activity. (A) Intracellular Ca²⁺ concentration was monitored in HEK293 cells overexpressing EYFP-D76N/D78N-Stim1 beginning in the presence of 1.8 mM extracellular Ca²⁺, followed at the time indicated by switch to a nominally Ca²⁺-free extracellular solution. Extracellular Ca²⁺ was then restored, and 100 µM ML-9 was added. In the continuous presence of ML-9, extracellular Ca²⁺ was again removed and restored. The trace represents the average response of all the cells measured on a single coverslip; representative of three independent experiments. (B) Time-lapse TIRFM was performed on a cell overexpressing EYFP-D76N/D78N-Stim1; 100 µM ML-9 was added at the time indicated in the fluorescence intensity profile (left panel). Right-hand panel: representative TIRFM images taken at the times indicated (i and ii) in the intensity profile. Representative of three independent experiments. (C) Confocal images of EYFP-D76N/D78N-Stim1-expressing cells in the absence (left panel) and presence (right panel) of 100 µM ML-9 in the presence of 1.8 mM extracellular Ca²⁺ throughout. Bars, 10 µm.

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