ML-9, an inhibitor of CCE, prevents the activation of AC8 by STIM1-mediated Ca²⁺ entry in HEK293 cells. A, confocal analysis of HEK293 cells coexpressing Orai1-myc, YFP-STIM1, and CFP-AC8 after store depletion with 200 nM TG in the presence and absence of ML9. Pretreatment with 100 μM ML9 (lower panels) limits translocation of STIM1 to the plasma membrane. Orai1-myc is shown in red, YFP-STIM1 in yellow, AC8-CFP in cyan, and 4,6-diamidino-2-phenylindole staining of the nucleus is in dark blue. B, dose-dependent effects of ML9 pretreatment on CCE in Fura-2 loaded HEK-AC8 cells. Each bar represents peak Ca²⁺ entry during CCE relative to control conditions (mean ± S.E.M., n values range from 43-98). Cells were incubated in Ca²⁺-free conditions in the presence of 0.2 μM TG and the presence or absence of ML-9 for 10 min, followed by the addition of 2 mM Ca²⁺. C, comparison of the effects of 10 and 100 μM ML9 on CCE in Fura2-loaded HEK-AC8 cells. Average traces ± S.E.M. of the Ca²⁺ entry occurring upon addition of extracellular Ca²⁺ are shown. D, ML-9 pretreatment prevents the activation of AC8 by CCE in a dose-dependent manner. HEK-AC8 cells expressing the Epac1-camps sensor were stimulated as described in A to measure [cAMP]_i. Average traces ± S.E.M. are shown. E, the peak [Ca²⁺]_i response and the initial rate of Ca²⁺ entry of the traces depicted in C were calculated. F, the peak [cAMP]_i response and the initial rate of AC8 activation of the traces depicted in D were quantified. ^***, p < 0.001 in a Student's t test.