Y 27632

BT549 and Hs578T cells were cultured in DMEM (Corning Cellgro) with 10% fetal bovine serum and 1% Penicillin/Streptomycin and maintained at 37°C in 5% CO₂. Y-27632 and Rho Activator II (CN03) were obtained from Cytoskeleton, Inc. The Rho Activator II drug concentration used for this study was determined based on the dose at which maximum stress fiber formation was observed, as recommended by the manufacturer. Primary antibodies used for immunoblotting and/or immunofluorescence were obtained from Cell Signaling Technology for P-MYPT1 (T853), Total-MYPT1, P-MLC (S19), Total-MLC, P-cofilin (S3), Total-cofilin and Acetyl (L-40) tubulin; Abcam for P-tau (S262) and detyrosinated-tubulin; and alpha-tubulin was obtained from Sigma.

Cells were seeded into transwell filters (8  μM pores; BD Biosciences) and incubated for 24 h in media (as above except FCS was reduced to 1%) containing 0.1–10 μM S1P (Sigma-Aldrich, Poole, UK) or vehicle (methanol). Cells were methanol-fixed then those on the upper surface of the membrane were removed using a cotton swab and the remainder were stained with Harris's hematoxylin (Sigma). Cells were viewed by light microscopy (Olympus BX41 microscope,×10 magnification); the number of cells in 6 fields of view were counted. In some experiments, the S1PR-1/3 inhibitor, FTY720 or pFTY720 (100 nM, Cayman Chemical Company, Ann Arbor, USA), the S1PR2 inhibitor, JTE-013 (100 nM, Tocris, Abingdon, UK) or the Rho kinase inhibitor, Y27632 (10 μM; Cytoskeleton Inc., USA) was added to cultures for 30 min before treatment with 100 nM S1P for a further 24 h. In experiments to assess the affect of 1,25-dihydroxyvitamin D (D₃; Sigma) on EVT migration, D₃ (1-10 nM) was added for 48 h or 72 h before incubation with 100 nM S1P for a further 24 h.