RASSF4 is a positive regulator of SOCE. (A) A diagram of the domain structure of RASSF4. The regions targeted by siRASSF4_N’ used in C, siRASSF4_C’ used in C, and siRASSF4 used in B are indicated. RA, RAS association. (B) Intracellular Ca²⁺ levels determined by analysis of Fura-2 fluorescence ratios in HeLa cells treated with a control siRNA (siControl), siSTIM1, or siRASSF4 and stimulated with 1 µM TG and 100 µM histamine (His). Shown are mean Fura-2 ratios ± SEM of >300 cells for each condition. Similar results were obtained from >10 independent experiments. (C) Fura-2 ratios of HeLa cells treated with the indicated siRNAs. Cells were stimulated with 1 µM TG, 100 µM His, and 2 mM EGTA; 2 mM Ca²⁺ was added 6 min after stimulation. Shown are mean Fura-2 ratios ± SEM derived from >1,000 cells for each condition across two independent experiments. (D) Fura-2 ratios of siRNA-treated HUVECs stimulated as described in C. Shown are mean Fura-2 ratios ± SEM derived from >500 cells for each condition across two independent experiments. (E) Fura-2 ratios of RPE-1 cells treated with the indicated siRNAs. Cells were stimulated with 1 µM TG and 2 mM EGTA; 2 mM Ca²⁺ was added 11.5 min after stimulation. Shown are mean Fura-2 ratios ± SEM of >280 cells for each condition. Similar results were obtained from three independent experiments. (F) Mn²⁺ influx measured by Fura-2 quenching in siRNA-treated HeLa cells stimulated with 1 µM TG. Shown are means ± SEM derived from >200 cells for each condition across two independent experiments. (G) HeLa cells were sequentially transfected with NFAT-YFP, and either siControl or siRASSF4 was treated with 1 µM TG for 10 min and scored for NFAT by fluorescence imaging. Percentage of cells with nuclear translocation of NFAT was calculated from 80–100 cells across three independent experiments. Means ± SEM are plotted. ***, P < 0.001. (H) HeLa cells were treated with siControl and a control vector, siControl and mCherry-STIM1-CT, or siRASSF4 and mCherry-STIM1-CT. Ca²⁺ was chelated using 2 mM EGTA, and then 2 mM Ca²⁺ was added to evaluate spontaneous Ca²⁺ influx. Mean Fura-2 ratios ± SEM of >500 cells across three independent experiments are plotted. (I) Fura-2 ratios in mCherry-STIM1–expressing HeLa cells treated with either siRASSF4 or siControl. Cells were stimulated as described in C. Shown are mean traces of >300 cells for each condition from a representative of three independent experiments.