Figure 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 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 (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 prior to addition for experiments performed as described in (B) for untreated controls (n = 6; 2 coverslips), or cells treated with 1 μM (n = 11; 3 coverslips), 10 μM (n = 11, 3 coverslips), 50 μM (n = 14, 3 coverslips) and 100 μM (n = 10, 3 coverslips) ML-9. Data are expressed as percent of untreated control ± SEM; * indicates significant difference compared to control (p < 0.001) based on one-way ANOVA. Scalebars = 10 μm.