Figure 1.

Store-operated Ca²⁺ entry in NIH 3T3 cells. (A) Store-operated Ca²⁺ entry (SOCE) in fibroblasts following passive endoplasmic reticulum (ER) Ca²⁺-store depletion with cyclopiazonic acid (CPA), an inhibitor of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). Cells grown on glass coverslips were loaded with Fura-2 and perifused with test solutions at 37 °C. Cells were initially bathed in standard extracellular solution (SES) containing 1 mM Ca²⁺ to establish a baseline. The bath solution was changed to a Ca²⁺-free SES (open bar; Ca²⁺-free), followed by Ca²⁺-free SES with 20 μM CPA to discharge and prevent refilling of the intracellular Ca²⁺ stores. SOCE was triggered by the subsequent application of SES with 1 mM Ca²⁺ (open bar; +Ca²⁺) in the continued presence of CPA; the rise of cytosolic [Ca²⁺] indicative of SOCE in single cells was measured using quantitative fluorescence microscopy and is expressed as the increase in the ratio of Fura-2 FI340/FI380. Data are expressed as the mean ± SEM (n = 67 cells). (B) Pharmacological analysis of SOCE. Cells were loaded with Fura-2 in SES and Ca²⁺ was measured with the FlexStation 96-well plate reader. The cells were incubated in a Ca²⁺-free SES containing CPA (20 μM) and the SOCE inhibitor or vehicle control. Application of GSK-7975A (GSK; 50 μM), SKF96365 hydrochloride (SKF; 10 μM), ML-9 (100 μM), or NAGly (30 μM) before the reintroduction of extracellular Ca²⁺ (open bar; +Ca²⁺) caused a significant reduction in the (C) normalized peak amplitude (Max FI335/FI375) and (D) area under the curve (AUC60) of the store-operated Ca²⁺ response. Graph data in (B) are plotted as the time-dependent change in the mean ± SEM of the fold change in the ratio of Relative FI335/FI375, averaged from 12 or more wells for each inhibitor from at least three independent experiments. In the box and whisker plots, the center solid line marks the median, small open square within the box depicts the mean, the ends of the box are the 25th and 75th quartiles, and whiskers are the minimum and maximum measured values. * p < 0.05 compared to vehicle control.