Depletion of BiP restores Y¹⁴³D-S1PR1 cell surface localization and endothelial barrier function. (A) Live cell TIRF images taken at 100× magnification from control siRNA (siSc) or BiP-depleted (siBiP) ECs transducing WT- or Y¹⁴³D-S1PR1. ECs were first transfected with siRNA and at 24 h and retransfected with indicated S1PR1 cDNA, and images were acquired after 48 h. Scale bar, 10 μm. (B) Quantification of S1PR1 surface expression (n = 7–10 cells/group) from experiments performed three times independently. ***, P < 0.0001 compared with WT-S1PR1 or siBiP plus Y¹⁴³D-S1PR1 by one-way ANOVA with two-tailed paired t test. (C) ECs transducing BiP siRNA and S1PR1 mutants as in A were stimulated with 1 μM S1P. Ca²⁺ release (first peak) or SOCE (second peak) was determined as in Fig. 6 G. (D) Mean ± SD of increase in Ca²⁺ (n = 8–10 cells/group) from experiments repeated three times. Basal Ca²⁺ (−) analyzed at 20 s; peak increase in Ca²⁺ from ER analyzed at 45 s while peak of SOCE was analyzed at 210 s. **, P < 0.001; ***, P < 0.0001 compared with unstimulated WT-S1PR1 or Y¹⁴³D-S1PR1; ##, P < 0.001; ###, P < 0.0001 compared with S1P-stimulated WT-S1PR1 by one-way ANOVA with two-tailed paired t test. (E) TEER in response to 1 μM S1P from ECs transducing BiP siRNA and S1PR1 mutants as in A. (F) Mean ± SD of TEER from experiments repeated three times. Basal TEER was calculated as mean between 10 and 30 min, while S1P-stimulated TEER was the mean between 1 and 1.5 h. ***, P < 0.0001 compared with unstimulated WT-S1PR1 or Y¹⁴³D-S1PR1; ###, P < 0.0001 compared with unstimulated siBiP plus WT-S1PR1 by one-way ANOVA with two-tailed paired t test. See also Figs. S4, C and D; and S5, A and B. A.U, arbitrary units.