D erythro s1p

Sphingosine 1-phosphate, and D-erythro (S1P) were purchased from Enzo Life Sciences (Plymouth Meeting, PA). C₁₇-Sphingosine 1-phosphate (C₁₇-S1P), lysophosphatidic acid (LPA), lysophosphatidylserine (LPS), lysophosphatidylinositol (LPI), and VPC23019 were purchased from Avanti Polar Lipids Inc (Alabaster, AL). Pertussis toxin (PTX) was obtained from Wako Pure Chemical Industries (Osaka, Japan).
S1P, LPA, LPS and LPI were dissolved in methanol. Just before use, the methanol was evaporated and the reagents were resolved in PBS containing 0.4% fatty acid-free BSA (Sigma-Aldrich Co., St. Louis, MO). C₁₇-S1P was dissolved in methanol. VPC23019 and PTX were dissolved in DMSO.

Huh7 HCC cell line was obtained from and authenticated by CellBank Australia, human dermal microvascular endothelial cells (HMEC-1) were originally from the American Type Culture Collection, while primary human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords authorized by the SLHD-HREC (#2019/ETH06859). Cells were maintained in a 5% CO₂ incubator using the previously detailed culture media [31 (link)–33 (link)]. Huh7 and HMEC-1 cells were tested mycoplasma-free. Short hairpin RNAs targeting SphK1 (#a or if not specified, 5ʹ-GCAGCTTCCTTGAACCATTAT-3ʹ; #b, 5ʹ-CGCTGTGCCTTAGTGTCTACT-3ʹ) were constructed in pLKO.1 lentiviral vector (Sigma-Aldrich). The lentivirus was generated in HEK293T cells using plasmids obtained from Dr Didier Trono through Addgene, including pMD2.G, pMDLg/pRRE and pRSV-Rev [34 (link)]. PF-543 and W146 used for cell culture were purchased from Cayman Chemical; MG-132 was from Calbiochem; VEGF-A was from R&D Systems; while D-erythro S1P was from Enzo Life Sciences.