DREAM regulates Ca²⁺ mobilization in MEG-01 cells and its regulatory function requires Ca²⁺ binding and PI3K-Iβ activity. Control or DREAM siRNA (#3 or #4) was transfected into MEG-01 cells. (A-B) After 48 hours, cell lysates were immunoblotted, followed by densitometry (arbitrary unit [AU], n = 4). (C) After 48 hours, cells were incubated with a Ca²⁺ dye and treated with 100 nM A23187, followed by measurement of cytosolic Ca²⁺ levels (n = 4). *P < .05 vs control after the Student t test. (D) A schematic of DREAM siRNA-resistant wt and EF-hand mutant DREAM. (E) DREAM siRNA #4 was transfected into MEG-01 cells. After 4 hours, cells were transfected with a vector expressing siRNA #4–resistant wt or EF-hand mutant DREAM. After 48 hours, DREAM overexpression was verified by immunoblotting (n = 4). (F) The cells described in panel E were used for Ca²⁺ assays. **P < .01 and ***P < .001 vs control after the Student t test (for siRNA#4) or wt DREAM-overexpressing cells (for EF-hand mutant groups) after ANOVA and the Tukey test. ##P < .01 between 2 groups. (G) Control or DREAM siRNA #4 was transfected into MEG-01 cells. After 48 hours, cells were incubated with a Ca²⁺ dye and treated with 0.05% DMSO, 25 µM LY294002 (LY), 0.1 µM wortmannin (Wort), 0.5 µM PIK75, or 0.5 µM TGX221. Ca²⁺ release was measured following stimulation with 100 nM A23187 (n = 4). (H) Control or DREAM knockdown MEG-01 cells were pretreated with vehicle or a PI3K inhibitor and stimulated with A23187 for 5 minutes. Immunoblotting was performed as described in “Methods” (n = 3). *P < .05, **P < .01, and ***P < .001 vs control (white bars in panel G) or DREAM siRNA-treated (gray bars in panel G) cells (vehicle group), or control siRNA-treated, stimulated cells (for panel H) after ANOVA and the Tukey test. #P < .05 and ###P < .001 between 2 groups.