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Fig. 4. ML-9 inhibits EYFP-Stim1 rearrangement. (A) Time-lapse TIRFM was performed on EYFP-Stim1-overexpressing HEK293 cells treated with 100 µM ML-9 or left untreated (control). The left panel shows TIRFM fluorescence intensity profiles for two control (black traces) and two ML-9-treated cells (red traces). Thapsigargin (Tg; 2 µM) was added in nominally Ca²⁺-free extracellular solution at the time indicated to deplete Ca²⁺ stores, and ML-9 was removed at the end of the experiment to demonstrate reversal of the ML-9 inhibition. The right-hand panel shows representative TIRFM images taken at the times indicated (i-iii) in the intensity profile. (B) TIRFM imaging was performed on three cells as described in panel A, but ML-9 (100 µM) was added after store depletion with thapsigargin. (C) The average baseline-subtracted TIRFM fluorescence intensity 5 minutes following ML-9 addition was divided by that just before addition for experiments performed as described in panel B for untreated controls (n=6; two coverslips), or cells treated with 1 µM (n=11; three coverslips), 10 µM (n=11, three coverslips), 50 µM (n=14, three coverslips) and 100 µM (n=10, three coverslips) ML-9. Data are expressed as the percentage of untreated control±s.e.m.; ^*, significant difference compared with control (P<0.001) based on one-way ANOVA. Bars, 10 µm.

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