Figure 7.

Ca²⁺ entry through Orai1 regulates AKT-dependent proliferation and TGF_β1 secretion in PS-1 human-activated PSCs. (A) Effect of extracellular Ca²⁺ decrease on AKT activation, 72 h post-proliferation, after overnight FBS starvation of no transfected cells. Then PS-1 cells were pre-treated for 30 min with 1.3 mM EGTA in order to reduce the extracellular Ca²⁺ concentration (0.1 mM Ca²⁺ ), with (+) and without (-) 10 min of 10% FBS stimulation. Representative Western blot of low extracellular Ca²⁺ concentration impact on AKT phosphorylation (Aa). AKT activation was evaluated as described previously, by the ratio of phosphorylated AKT form/total AKT protein (1.4 mM Ca²⁺ +10 min FBS: 8.5 ± 2.52-fold, 0.1 mM Ca²⁺ + 10 min FBS: 2.92 ± 0.46-fold, N = 3, * p < 0.05, NS, two-way ANOVA followed by Bonferroni post hoc test, (Ab). Values were first normalized to the referent protein GAPDH and then to the 0% FBS + 1.4 mM Ca²⁺ condition, reported as mean ± SEM. (B) Evaluation of siOrai1 transfected PS-1 cells’ proliferation after 72 h of 1.3 mM EGTA treatment by MTT assay. (C) Impact of Orai1 knocked-down cells, treated with low Ca²⁺ condition, on cell survival, assessed by Trypan blue assay. Cell mortality was calculated using the formula: % of cell death = number of dead cells/number of total cells, reported as mean ± SEM for each condition. (D) Similarly, TGF_β1 secretion was measured in Orai1 knocked-down PS-1 cells, in the presence of low-Ca²⁺ conditions, by ELISA assay. All values were normalized to 1.4 mM Ca²⁺ control condition (except for the mortality rate) and reported as mean ± SEM (*** p < 0.001, * p < 0.05, NS, N = 3, one-way ANOVA followed by Bonferroni multiple comparison test).