Fig. 2

NRP2 modulates angiogenesis by promoting HUVEC migration via a VEGF/VEGFR2-independent pathway. a HUVECs were cultured in the presence or absence of conditioned medium from BON cells (treatment and control, respectively) and then transfected with a vector control or NRP2 overexpression plasmid before they were seeded for the capillary tube formation assay. Representative images at 4, 12 and 24 h after plating are shown. b Quantification of the number of complete and broken tubes at 6 h from a representative experiment. Data are shown as the mean ± SD of three independent experiments. *P ≤ 0.05 by Student’s t test. c HUVECs were treated with conditioned medium from BON cells for 24 h and then transfected with a vector or NRP2 overexpression plasmid before they were subjected to a CCK8 assay. d After HUVECs were cultured in the presence or absence of conditioned medium from BON cells (treatment and control, respectively) and transduced with the NRP2-overexpressing plasmid, flow cytometry was performed to assess apoptosis. e Representative images for the wound-healing assay at 0, 24 and 48 h after scratching for the 4 different cell groups (HUVECs with or without NRP2 overexpression cultured in the presence or absence of conditioned medium from BON cells). f Quantification of the healing rate at 48 h after wound-healing assays in HUVECs cultured in the presence or absence of conditioned medium from BON cells followed by transfection with empty vector or NRP2 plasmid. The data are shown as the means ± SD of three independent experiments. ***P ≤ 0.001 by Student’s t test. g HUVECs were transfected with empty vector or an NRP2 overexpression plasmid and then treated with the VEGFR2-specific inhibitor KI8751. Western blotting assays were performed to determine the levels of VEGFR2 phosphorylation at Tyr951 as well as the total protein levels of VEGFR2, CD31, CD34 and GAPDH. h Control and NRP2-overexpressing HUVECs were treated with KI8751 and PBS and evaluated for tube formation. i After HUVECs were cultured in the presence or absence of conditioned medium from BON cells for 24 h, they were transfected with a vector or NRP2 overexpression plasmid. These cells were subsequently treated with PBS (control) or KI8751, and a wound-healing assay was performed. Representative image of three independent experiments is shown. j Qualification of the wound-healing rate at 48 h in HUVEC-vector or HUVEC-NRP2 cells treated with PBS or KI8751. The data are shown as the mean ± SD of three independent experiments. **P ≤ 0.01 by Student’s t test. k After HUVECs were transfected with empty vector or an NRP2 overexpression plasmid, they were treated with PBS or KI8751 and subjected to the CCK8 assay at 24, 48 and 72 h after treatment. l Flow cytometry assay was performed in the 4 cell groups described in Fig. 2k