S1P- and LPA-stimulated MT1-MMP–dependent morphogenic differentiation and EC migration involve heterotrimeric G_i protein. (A) (i) After adhesion of HUVECs to Matrigel, PTX (10 ng/mL) was added 30 minutes before the addition of either 1 μM S1P, 10 μM LPA, or 50 ng/mL VEGF. Original magnification, × 50. (ii) Formation of capillary-like structures was recorded and quantified using a computer-based program. Data are expressed as x-fold induction ± SD of nonstimulated control. Results are the means of 2 independent experiments. (B) HUVECs were allowed to attach to filters and were pretreated with PTX (10 ng/mL) for 30 minutes. The medium in the lower chambers was then replaced with serum-free media containing either 1 μM S1P, 10 μM LPA, or 50 ng/mL VEGF and incubated for 3 hours. Cell migration was quantified using a computer-based program and data are expressed as x-fold induction ± SD of nonstimulated control. Results are the means of 2 independent experiments. (C) BAECs were transfected with empty vector (pcDNA3.1) or with MT1-MMP construct and allowed to recover for 40 hours. (i) Transfected cells were allowed to adhere to Matrigel and PTX (10 ng/mL) was added 30 minutes before the addition of 1 μM S1P, 10 μM LPA, or 50 ng/mL VEGF. Original magnification, × 50. (ii) Formation of capillary-like structures was recorded after 6 hours and quantified using a computer-based program. Data are expressed as x-fold induction ± SD of nonstimulated control. Results are the means of 2 independent experiments. (D) Transfected BAECs were allowed to attach to filters and were then pretreated with PTX (10 ng/mL, 30 minutes). The medium in the lower chambers was replaced with serum-free medium containing either 1 μM S1P, 10 μM LPA, or 50 ng/mL VEGF and incubated for 3 hours. Cell migration was quantified and data are expressed as x-fold induction ± SD of nonstimulated control. Results are the means of 2 independent experiments.