Fig. 4. Microtubule depolymerization inhibits SOCE. (A) Wild-type HEK 293 cells were treated with 100 µM colchicine for 20 minutes (red trace) or left untreated (control; black trace), and thapsigargin (Tg; 2 µM) was then added in the presence of 1.8 mM extracellular Ca²⁺. The intracellular Ca²⁺ concentration was monitored throughout the experiments, and SOCE became activated soon after Tg addition, as evidenced by the second peak in Ca²⁺ concentration, and remained active based on the sustained elevation of intracellular Ca²⁺ above baseline. Each trace represents the average response of all cells on a single coverslip (20-30 cells). (B) The average difference between the 340/380 value 15 minutes following Tg addition (sustained SOCE) and the 340/380 value just before Tg addition (baseline) was calculated for untreated control (n=150, three coverslips) and colchicine-treated (n=145, three coverslips) cells for experiments performed as described in (A). (C) SOCE was analyzed as described in (A) for cells treated for 20 minutes with 10 µM nocodazole (NZL; red trace) and for control cells treated with 0.1% DMSO (black trace). Because nocodazole treatment alone caused some store depletion and activation of SOCE (see Fig. 7), nocodazole and DMSO treatments were performed in a nominally Ca²⁺-free extracellular solution. (D) The average difference between the 340/380 value 15 minutes following Tg addition (sustained SOCE) and the 340/380 value just before Tg addition (baseline) was calculated for DMSO-treated (n=122, three coverslips) and nocodazole-treated (n=125, three coverslips) cells for experiments performed as described in (C). The data are reported as the mean±s.e.m.; the P values are based on t-tests.