Imatinib

HDMECs (Promocell) were cultured in MV2 media with supplements (Promocell). HUVECs (Promocell) were cultured in EBM2 media (Lonza) containing 10% FBS (Life Technologies), 300 µg/ml EC growth supplement (ECGS; Sigma-Aldrich), and 1 µg/ml hydrocortisone (Sigma-Aldrich). Cells were transfected with lipofectamine RNAIMAX using SMARTpool siRNA targeting NRP1, VEGFR2, or ABL1 (Dharmacon), or Silencer negative control siRNA (Applied Biosystems). For overexpression studies, HDMECs were transfected with Lipofectamine according to the manufacturer’s protocol. In some experiments, cells were incubated with 10 µM Imatinib (Cambridge Bioscience), a concentration known to effectively target ABL1 kinase (Chislock et al., 2013 (link)). In some experiments, cells were stimulated with 50 ng/ml VEGF165 for the indicated times. Primary MLECs were isolated from mice between 1 and 2 mo of age by magnetic-activated cell sorting (MACS) with PECAM antibodies (BD). MLECs were cultured on 10 µg/ml FN in DMEM-GlutaMAX supplemented with 20% FBS, nonessential amino acids (Life Technologies), and ECGS. MLECs from Nrp1^fl/fl conditional null mice were infected with adenovirus-expressing CRE recombinase or with control virus–expressing GFP, incubated for 24 h in culture media, washed in PBS, and cultured in fresh media for 48 h before migration and immunoblotting experiments.