Vegf a

For primary CD4⁺ T-cell isolation, PBMCs were acquired utilizing Ficoll Paque Plus (GE Healthcare, USA) gradient cell separation according to the manufacturer’s instructions. Next, the CD4⁺ T Cell Isolation Kit, human (Miltenyi Biotec, Germany) was utilized to isolate CD4⁺ T cells. Then, the cells were resuspended at 1 × 10⁶ cells/mL and cultured in advanced RPMI1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% 2-mercaptoethanol (2-ME; Gibco) and 1% penicillin–streptomycin (Gibco) in 48-well plates precoated with 2 μg/mL anti-human CD3 antibody (BioLegend, USA). For VEGF-R-related assays, after incubation with 5 μg/mL anti-VEGF-R1 antibody (R&D Systems, USA) and/or 10 μg/mL anti-VEGF-R2 antibody (Abcam, USA) for 1 h, CD4⁺ T cells were treated with blank (CON) or 15 ng/mL VEGF-A (R&D Systems, USA) for 2 days respectively. GolgiPlug Protein Transport Inhibitor (BD Bioscience, USA) was administered for the last 6 h to detect the cytotoxic molecules. Regarding AKT/mTOR inhibition assays, CD4⁺ T cells were treated with blank (CON), 15 ng/mL VEGF-A, VEGF-A + 10 μM MK-2206 2HCl (AKT inhibitor, MedChemExpress, USA), VEGF-A + 100 nM rapamycin (mTOR inhibitor, Selleck, China) or VEGF-A + 10 μM MK-2206 2HCl + 10 μM MHY1485 (mTOR activator, MedChemExpress, USA) for 2 days in the presence of GolgiPlug Protein Transport Inhibitor for the last 6 h.

After thawing at 4 °C overnight, we plated Matrigel (Corning, Cat# 356231) in 96-well culture plates and incubated at 37 °C to polymerize for at least 30 min. Transfected siRNA or plasmid ligament cells were digested by trypsin and resuspended in endothelial cell medium (ECM, 1001, ScienCell) supplemented with 5% FBS, 1% endothelial cell growth factors, and 1% penicillin/streptomycin. We then seeded (1 × 10⁴ cells/well) on polymerized Matrigel. Sorafenib Tosylate (Cat# HY-10201A), Imatinib (Cat# HY-50946), recombinant human PDGF-BB (Cat# HY-P7055), VEGFA (Cat# HY-P7110A), VEGFA monoclonal antibody Bevacizumab (Cat# HY-P9906), and isotype control antibody (Cat# HY-P99001) were obtained from MedChemExpress (MCE). We observed and imaged the tube-like structures by microscopy after incubation at 37 °C for 4–6 h. The total length of tube-like structures was calculated with the help of ImageJ software (ImageJ; National Institutes of Health).