Effect of inhibition of Gi on cytosolic calcium and barrier function in response to S1P. (A and B) After 48 h after transfection, HEK cells transducing vector (GFP), GFP-tagged WT-S1PR1 or Y¹⁴³D S1PR1 cDNAs were left untreated or pretreated with PTX (50 μM) for 2 h. Cells were then loaded with Fura-2 AM for 15 min. Ratiometric analysis of [Ca²⁺]_i was then performed after stimulation with 1 μM S1P. A shows representative traces, whereas B shows individual data points (n = 8–10 cells/group). Basal Ca²⁺ (−) was analyzed at 45 s; peak increase in Ca²⁺ was analyzed at 90 s after S1P stimulation in control or PTX-treated cells. Note that only GFP-expressing cells were chosen to assess [Ca²⁺]_i release in all experiments. **, P < 0.001; ***, P < 0.0001 compared with unstimulated cells transfected with vector or S1PR1-GFP; ###, P < 0.0001 compared with S1PR1-GFP transfected cells after S1P stimulation by one-way ANOVA with two-tailed paired t test. (C and D) HUVECs seeded on gold-plated electrodes were transfected with S1PR1 or Y¹⁴³D-S1PR1 cDNAs. Cells were pretreated with or without PTX (50 μM) for 2 h. TEER in real time was determined in response to 1 μM S1P. An individual TEER trace is shown in C, while D shows the quantification of the TEER from experiments that were repeated at least two times. Basal TEER in control or PTX-treated ECs was quantified as the mean between 10 and 20 min, whereas TEER after S1P addition in these ECs was calculated as the mean between 2 and 3 h. ***, P < 0.0001 compared with unstimulated cells transfected with S1PR1-GFP; ###, P < 0.0001 compared with S1PR1-GFP–transfected cells treated with PTX and S1P by one-way ANOVA with two-tailed paired t test.