Fig. 1.

Effect of eicosapentaenoic acid (EPA) on cytoplasmic and endoplasmic reticulum (ER) Ca²⁺, and on the translocation of YFP-STIM1 in NCM460 cells. A: change in intracellular Ca²⁺ in response to 50 μM EPA in the absence and presence of extracellular Ca²⁺ compared with signal elicited by carbachol (“carb”; 100 μM) as measured with fura 2. Error bars indicate ± SE. B: the response to 50 μM EPA (shown here in Ca²⁺-free solutions) was highly variable when assessed at the single cell level. C: experiments with the low-affinity Ca²⁺ indicator mag-fura 2 to measure free Ca²⁺ in the ER lumen in permeabilized cells. The mag-fura 2 ratio decreased, indicating reduction of free Ca²⁺ in the lumen of the ER in response to inositol-1,4,5-trisphosphate (InsP₃) and EPA. D: as in C, the mag-fura 2 ratio change following InsP₃ treatment was not further lowered by subsequent addition of EPA. Ionomycin (5 μM) yielded an additional loss of stored Ca²⁺. E: bottom, time course of YFP-STIM1 translocation to the cell surface following treatment with 50 μM EPA and 5 μM ionomycin as measured using total internal reflection fluorescence (TIRF) microscopy. Intensity was measured from the regions encompassing the entire two cells; top, a-d: TIRF images of punctae at indicated time points; e: conventional epifluorescence image of same cells showing cellular outline (scale bar = 15 μm). Data typical of results from n = 7 cells in 5 independent experiments.