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. 2021 Nov 11;12:6537. doi: 10.1038/s41467-021-26900-w

Fig. 4. Insulin stimulates cell-surface abundance of Orai1 via VAMP2-associated exocytosis in a PI3K-dependent pathway.

Fig. 4

a Effect of insulin on the CRAC current in HEK293FT cells heterologously co-expressing 3xflag-mCherry-tagged Orai1 and YFP-tagged STIM1. Inset shows current–voltage relationship of the relative (Rel.) CRAC current with or without insulin treatment. b Summary of the Orai1 current density at −100 mV in a. n = 10 for Vehicle and Insulin-treated cells. c Representative traces of SOCE in cultured mouse podocytes from mice treated with vehicle (HCl) or insulin (100 nM). Inset shows the slope of the scaled-up relative Ca2+ rise kinetics between 600 and 700 s. d Time-window of insulin stimulation on store-operated Ca2+ entry (SOCE) in podocytes. n = 28, 20, 21, 27, and 19 cells, respectively. e Biotinylation assay showing the effect of insulin (100 nM, 10 min, or 1 h) on the cell-surface abundance of Orai1. f Quantification of the surface Orai1 by insulin in e. n = 3 independent experiments. g Effect of the knockdown of VAMP2 by siRNA on insulin-stimulated Orai1 cell-surface expression in podocytes. h Quantification of the results in g. n = 3 independent experiments. i Representative fura-2 ratio showing the effect of VAMP2 siRNA knockdown on insulin-stimulated SOCE. j Summary of the SOCE in i. n = 30 and 43 cells for Ctrl Oligo and siVAMP2, respectively. k Biotinylation assay representing the effects of PI3K inhibitors (wortmannin, WMN; 100 nM and LY294002, LY; 10 μM, treatment for 1 h) on the insulin-stimulated cell-surface abundance of Orai1 in cultured mouse podocytes. Lower panel: immunoblotting analysis validating PI3K-Akt pathway response to insulin stimulation (100 nM, 15 min). Akt phosphorylation at serine 473 (p-Akt (S)) and at threonine 308 (p-Akt (T)). l Quantification of the k. n = 3 independent experiments. m Representative SOCE traces showing the effect of PI3K inhibitors on insulin-induced SOCE. n Summary of the SOCE in m. n = 28, 29, and 33 cells per each group. Data are expressed as mean ± SEM and analyzed by one-way ANOVA followed by Dunnett’s (d, f, n) or Tukey’s (h, l) multiple comparisons test, and unpaired two-tailed Student’s t-test (b, j). ****p < 0.0001; ns not significant.