Figure 1.
Orai3 is overexpressed in lung adenocarcinoma and correlated with high tumor grade.
A: Representative examples of Orai3 expression in cancerous and matched non-tumoral human lung tissues, as assessed by immunohistochemistry. Original magnification: ×400. Insert negative control obtained by omitting the primary antibody. B, quantitative analysis of Orai3 staining score in tumoral and adjacent non-tumoral lung tissue (n = 60, *** p<0.001, Mann-Withney test). C, Typical examples of Orai3 immunoreactivity in grade 2 lung cancer tissue (weak staining) and in grade 3 lung adenocarcinoma tissue (strong staining) at a magnification of ×400. Inserts negative control obtained by omitting the primary antibody. D, quantification of Orai3 staining score in grade 1–2 versus grade 3 lung cancer (n = 40, p = 0.032, Mann-Withney test).
Table 1.
Clinical characteristics of patients who underwent pulmonary resection for adenocarcinoma.
Figure 2.
Thapsigargin (TG, 1 µM) applied to NCI-H23 cells (A) or NCI-H460 (B) in 0 mM Ca2+ solution (0Ca) induced an increase of F350/F380 fluorescence. Re-introduction of 5 mM Ca2+ solution (5Ca) induced stable increase in Ca2+ signal, a characteristic of SOCE. Gd3+ applied in 5Ca solution induced inhibition of this Ca2+ signal. (p<0.001, n = 25 for NCI-H23 and n = 28 for NCI-H460, Mann-Withney test).
Figure 3.
Silencing of Orai1 and Orai2 does not change SOC entry in the two cell lines.
Expression of Orai1, Orai2, Orai3, Stim1, and Stim2 estimated in NCI-H23 (A) and NCI-H460 (B) cells using semi-quantitative RT-PCR. si-Orai1 has no effect on SOCE in NCI-H23 (C, n = 125) and NCI-H460 (D, n = 150). Silencing Orai2 (si-Orai2) induces increase in SOCE in NCI-H23 (E, n = 125, p<0.001, Mann-Withney test), but has no effect on SOCE in NCI-H460 (F, n = 125) cells.
Figure 4.
Effect of Orai3 knockdown on SOC entry.
The effect of siRNA against Orai3 on thapsigargin-activated SOCE in (A) NCI-H23 cells (n = 25 cells for each condition) and (C) NCI-H460 cells (n = 27 cells for each condition). Down-regulation of Orai3 expression produced a significant reduction of basal calcium concentration. [Ca2+]i is measured after 1 minute of standard solution extracellular perfusion. B–D) Quantitative analysis of SOCE in NCI-H23 cells (B, n = 25) and NCI-H460 cells (D, n = 27). The values presented as mean ± SEM. (***p<0.001, Mann-Withney test). Perfusion of 50 µM 2-APB transiently potentiates SOCE in NCI-H23 (E, n = 45) and NCI-H460 cells (F, n = 54). The values presented as mean ± SEM. (***p<0.001, Mann-Withney test).
Figure 5.
Down regulation of Orai3 reduced cell proliferation and arrested cells in G0/G1 phase.
A, Effect of Orai3-knockdown on NCI-H23 cell viability (*** p<0.001, t-test). B, The same experiments were carried on NCI-H460 cells (*** p<0.001, t-test). Cell viability was measured 72-h post-transfection and was normalized as a percentage of the control and results were expressed as mean ± SEM of three independent experiments. C–D, a cell-cycle distribution of NCI-H23 (C) and NCI-H460 (D) cells transfected with si-Orai3 or si-CTL carried out by flow cytometry of the cells stained with Propidium Iodide. Inserts: traces representing distribution of the cells in the cell cycle. Asterisks denote statistical significance as compared to control cells; ***p<0.001 of three independent experiments (t-test). E–F, An apoptosis assay carried out by Annexin V staining. Orai3 silencing failed to affect NCI-H23 (E) and NCI-H460 (F) apoptosis.
Figure 6.
Silencing of Orai3 reduced the up-regulation of cyclin and CDK expression protein levels induced by serum.
Cells were transfected by si-Orai3 or si-CTL during 72-h and the expression levels of cell cycle protein were analyzed by Western blotting. A, Representative immunoblots of the expression of cyclin D1, E, Cdk4 and Cdk2 in NCI-H23 cells transfected with si-CTL or si-Orai3. B, Protein levels were quantified and normalized to actin. The indicated values are mean ± SEM of 3 independent experiments, *p<0.05, Mann-Withney test. C, Representative immunoblots of the effect of si-Orai3 on cyclin D1, cyclin E, Cdk4 and Cdk2 expression in NCI-H460 cells. D, Protein levels were quantified and normalized to actin. The indicated values are the mean ± SEM of 3 independent experiments, *p<0.05, Mann-Withney test.
Figure 7.
Effect of si-Orai3 on thapsigargin and serum induced AKT phosphorylation.
A, B, Representative western blotting of P-Akt and Akt proteins in NCI-H23 (A) and NCI-H460 cells (B) transfected with si-CTL or si-Orai3. Each siRNA was tested in 0% serum (FCS), 0% FCS+1 µM thapsigargin (TG), 10% FCS, and 10% FCS plus 1 µM TG. The quantification of the ratio P-Akt/Akt in NCI-H23 and NCI-H460 cells using densitometric analyses is shown in C and D (n = 2, p<0.05, One Way Anova on Ranks).