VEGF induces VEGFR2 signaling in and mediates ALL cell migration through CNS microvascular ECs. (A) VEGFR2 expression in CNS microvascular ECs (bEnd.3) but not NALM-6 BCP ALL cells. Western blot analysis; α-tubulin shows loading of equal amounts of protein. (B) Induction of VEGFR2 downstream signaling by AKT473⁻ phosphorylation and SRC416⁻ (western blot analysis) on VEGF exposure (100 ng/mL) at indicated increasing time points. (C) Migration of NALM-6 leukemia cells (red) through CNS microvascular ECs (blue). Increased transendothelial migration in the presence of VEGF (D) or of VEGF-overexpressing NALM-6 cells (E). Decreased migration on VEGF capture by bevacizumab (F) or of NALM-6 cells with VEGF downregulation (G). Data points represent numbers of migrated cells per well (n = 21, VEGF 0 ng/mL; n = 24, VEGF 50 ng/mL; n = 23, VEGF 100 ng/mL; n = 24, green fluorescent protein [GFP] control [ctrl] VEGF overexpression; n = 23, VEGF overexpression; n = 23, bevacizumab 0 ng/mL; n = 24, bevacizumab 25, 50, and 100 ng/mL; n = 24, GFP ctrl VEGF knockdown; n = 23, VEGF microRNA [miRNA]); bars indicate median values; significance by 2-tailed Mann-Whitney U test; P values as indicated in graphs.