Overexpression of S1P1 in HUVECs

HUVECs (Allcells, Shanghai, China) were cultured in HUVEC medium (HUVEC-004; Allcells) as previously described (Zeng et al., 2019 (link)). Cells (passages three to six) were maintained at 37°C with 5% CO₂ in a humidified incubator. Lentivirus infections of miR-9 mimic (5′-TCTTTGGTTATCTAGCTGTATGA-3′) and negative control (NC; 5′-UUGUACUACACAAAAGUACUG-3′; B04001; GenePharma, Shanghai, China) were performed as previously described (Zeng et al., 2019 (link)). cDNAs for S1P₁ were ligated into plasmid pcDNA3.1. For overexpression of S1P₁, cells were transfected with pcDNA3.1 vector expressing S1P₁ using Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, United States). Transfected cells after 48 h were used for the subsequent experiments. Recombinant human VEGF 165 Protein (50 ng/mL; hVEGF; 293-VE, R&D system, Minneapolis, MN, United States) was used to test the migration, invasion and in vitro angiogenesis of cells overexpressing S1P₁.