Table 1.
Primers set used for the quantitative PCR analysis.
Figure 1.
STIM1 and STIM2 are expressed in BAECs and contribute to SOCE.
A) Cells were transfected with siCtrl, siSTIM1 or siSTIM2. After 48 h, cells were lysed and proteins were resolved by SDS-PAGE and identified by Western blot using selective antibodies against STIM1, STIM2 or actin (NT indicates non transfected cells). B) BAECs were loaded with fura-2/AM and imaged using an Olympus IX71 microscope (40× oil immersion objective) coupled to a MetaFluor imaging system for the recording of the intracellular Ca2+ concentration. In a nominally free Ca2+ medium, cells were treated with 1 µM TG to deplete their Ca2+ store and, once the Ca2+ concentration had stabilized, 1.8 mM Ca2+ was added to the medium to induce Ca2+ entry. The figure shows average traces from cells (>75 cells/condition) transfected with siCtrl (black line), siSTIM1 (dashed line) or siSTIM2 (gray line). C) Average Ca2+ increase (peak amplitude) after treatment with TG and subsequent Ca2+ entry (mean ± SD of results obtained from 3–4 independent experiments, *p<0.05). D) Total RNA was extracted from transfected cells (48 h post-transfection) and subjected to a qPCR analysis using specific primers for STIM1 and STIM2 to evaluate their relative level of encoding mRNAs. The results represent the mean ± SD of three independent experiments.
Figure 2.
STIM1 and IP3R-1 are widely distributed throughout the endoplasmic reticulum in BAECs.
A) BAECs were grown on cover glasses, fixed with methanol and incubated with mouse anti-STIM1 and rabbit anti-IP3R-1 antibodies. Fluorescent staining was obtained with AlexaFluor 594-conjugated (STIM1, left panel) and AlexaFluor 488-conjugated (IP3R-1, center panel) secondary antibodies. The right panel represents the overlay of these images. The results are representative of three independent experiments performed on different cells preparations.
Figure 3.
STIM1 and STIM2 interact with IP3R-1.
A) BAECs were solubilized in 1% Triton X-100 and the lysate was fractionated into samples that were immunoprecipitated with isoform-specific anti-STIM antibodies or, as control conditions, with IgG antibodies (IgG) or exclusively with protein-A/G agarose beads (A/G). The resulting immune complexes were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with an isoform-specific anti-IP3R-1 antibody as indicated on the left side of the blot. B) BAECs lysate was immunoprecipitated with anti-IP3R-1 antibody and the blot was revealed with an anti-STIM1 or anti-STIM2 antibodies as indicated. These results are representative of at least three independent experiments performed with different cells preparations.
Figure 4.
The knockdown of STIM1 dampens the IP3R-dependent agonist-induced intracellular Ca2+ release in BAECs.
BAECs were loaded with fura-2/AM and imaged using an Olympus IX71 microscope (40× oil immersion objective) coupled to a MetaFluor imaging system for the recording of intracellular Ca2+ concentration. (A and B) Average traces from cells (>75 cells/condition) transfected with siCtrl (black line), siSTIM1 (dashed line) or siSTIM2 (gray line) stimulated with 100 nM ATP (A) or 5 nM BK (B), in a nominally free Ca2+ medium. (C and D) Average Ca2+ releases (mean ± SD of results obtained from 4–6 independent experiments) induced by increasing concentrations of ATP (C) or BK (D). (E and F) Same data as in C and D expressed as the percentage of the maximal response under each condition. * indicates that the results are significantly different from those obtained with cells transfected with siCtrl.