Figure 1.

Essential role of gene CG9126 (Stim) in SOC influx in Drosophila S2 cells. (A) Drosophila SOC influx measured in a fluorimeter. Basal-subtracted fluo-4 fluorescence in relative fluorescence units (RFUs) from Drosophila S2 cells in a 96-well plate. Cells were initially in Ca²⁺-free solution (Ca0). Bars indicate addition of TG (1 μM, solid line) or vehicle (dotted line), followed by 2 mM Ca²⁺ (Ca2). The TG-independent response can be explained by partial store depletion during exposure to Ca²⁺-free solution or possible damage to some cells. Traces are averages of recordings from four individual wells. (B) Development of an end point assay for screening gene candidates. Cells were treated as described in A and placed in a fluorimeter 3 min after adding 2 mM Ca²⁺. Pre-incubating cells with 20 μM 2-APB reduced TG-dependent Ca²⁺ entry significantly (P < 5 × 10⁻⁶: unpaired t test) and to a greater extent than the TG-independent Ca²⁺ entry (P < 5 × 10⁻⁶: unpaired t test); n = 24 for each treatment group. (C) Treatment of Drosophila S2 cells with Stim dsRNA inhibits SOC influx by 90% (P < 10⁻⁵; unpaired t test compared with control mock-treated cells). Data represent basal-subtracted RFUs divided by maximal fluorescence (F_max) to normalize for cell number. The TG-dependent Ca²⁺ signal can be obtained by subtracting the average TG-independent signal from the Ca²⁺ signal after treatment with TG. Knockdown of Stim also inhibited the TG-independent Ca²⁺ signal (vehicle) by <10%, albeit significantly (P < 10⁻⁴, unpaired t test). (D) mRNA reduction in Stim dsRNA-treated cells. RNA was isolated from mock-treated cells or cells treated with a dsRNA specific to Stim. RT-PCR analysis was performed using gene-specific primers to Stim or to a control gene, presenilin (PSN). (E) Suppression of other candidate genes did not markedly inhibit TG-induced Ca²⁺ influx. Cells were mock treated or treated with dsRNA specific for CG11059, CG1560, trp-l, CG8743, or Stim for 5 d. Ca²⁺ influx was measured after pretreatment with TG. Data represent basal-subtracted RFUs divided by F_max to normalize for cell number. (F) Knockdown of CG8743 or CG2165 elevates basal intracellular Ca²⁺ levels. Cells were mock treated (control) or treated with dsRNA specific for CG8743 or CG2165 for 5 d. Data represents basal RFUs divided by the maximum fluorescence, F_max, to normalize to cell number (P < 0.01, one-way ANOVA, Dunnett's multiple comparison test compared with control mock-treated cells). (B, C, E, and F) Error bars represent means ± SD.