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. 2021 May 15;13(10):2395. doi: 10.3390/cancers13102395

Figure 3.

Figure 3

Orai1 mediates AKT activation but not ERK1/2 nor SMAD2 phosphorylation in human-activated PSCs. (A) Involvement of Orai1 in AKT phosphorylation in PS-1 cells. Representative Western blot showing the effect of Orai1 inhibition after FBS starvation of transfected cells overnight. Cells were then stimulated 10 min with FBS to evaluate the impact of Orai1 on AKT activation (Aa). AKT phosphorylation was quantified by the ratio of phosphorylated AKT form/total AKT protein (siCtrl + 10 min FBS: 5.63 ± 0.99-fold, siOrai1 + 10 min FBS: 2.18 ± 0.69-fold, (Ab). (B) Assessment of ERK1/2 activation after Orai1 knocking down in PS-1 cells. Representative Western blot showing the effect of Orai1 silencing on ERK1/2 activation, using the protocol described above (Ba). ERK1/2 phosphorylation was quantified by the ratio of phosphorylated ERK1/2 form/total ERK1/2 protein (Bb). (C) Evaluation of Orai1 silencing on SMAD2 phosphorylation. Representative Western blot showing the effect of siOrai1 transfected cells on SMAD2 activation (Ca), with the quantification using the ratio of phosphorylated SMAD2 form/total SMAD2 protein (Cb). All values were first normalized to the referent protein GAPDH and then to the 0% FBS control condition. All experiments were realized 72 h post-transfection. Values were reported as mean ± SEM (** p < 0.01, NS, at least N = 3, two-way ANOVA followed by Bonferroni post hoc test).