Modulating SphK1 in Huh7 and HMEC-1 Cells

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.