LPA/LPAR1 attenuates S1PR1-mediated barrier function. (A–D) HUVECs were analyzed for barrier function by real-time measurement of transendothelial electrical resistance (TEER) in the absence (A and B) or presence (C and D) of doxycycline (Dox), which can induce LPAR1 expression by the Tet-On system. 1 d after seeding, the cells were starved with 0.5% charcoal-treated FBS in the absence (A and C) or presence (B and D) of 1 µM Ki16425. At time 0, 100 nM AUY954 (blue), LPA (orange), AUY954 with LPA (dark green), or vehicle (black) was added. Data are from n = 3 independent experiments and expressed as mean ± SD. P values were determined by two-way ANOVA followed by Sidak’s multiple comparisons test comparing “AUY954 + LPA” to AUY954 alone; *, P ≤ 0.0001. (E–H) HUVECs expressing LPAR1 were starved with 0.5% charcoal–treated FBS for 2 h and then treated with 100 nM AUY954 and/or LPA for 30 min. Cells were fixed and stained for VE-cadherin (red) and p-MLC (green). F-actin and nuclei were stained with phalloidin (white) and DAPI (blue), respectively. Arrowheads indicate intercellular gaps. Bars, 20 µm. (I) Quantification of VE-cadherin–positive area in above confocal images expressed as mean ± SD. P value was determined by one-way ANOVA followed by Holm–Sidak’s multiple comparisons test; **, P ≤ 0.0021; *, P ≤ 0.0332. (J) Quantification of junctional length in above confocal images expressed as mean ± SD. P value was determined by two-way ANOVA followed by Tukey’s multiple comparisons test; ***, P ≤ 0.0002; **, P ≤ 0.0021; *, P < 0.0332. ns, not significant. AUY, AUY954.