NRP-1 mediates VPF/VEGF-induced EC survival; the C-terminal three amino acids of NRP-1 are essential for this function. Quantitative determination of apoptotic nuclei in PAEC (n = 8). TUNEL assay was performed in apoptosis-induced PAEC and PAEC/NRP-1 with or with out 10ng/ml VPF/VEGF stimulation for 48 hrs to assess whether NRP-1 mediates PAEC survival. b. FACS apoptosis analysis to determine the percentage of apoptotic PAEC. c. Quantitative determination of apoptotic nuclei in HUVEC (n = 8. n: The number of counted field. All are same in this study). EGNP-1 mediates HUVEC survival; the C-terminal three amino acids of NRP-1 are essential for this function. TUNEL assay was performed in apoptosis-induced HUVEC transfected with EGNP-1 or EGNP-1&detla;SEA and stimulated with or without 10ng/ml EGF. d. FACS apoptosis analysis to determine the percentage of apoptotic HUVEC. All the experiments at least were repeated three times and duplicated-wells were used for each experiment. *p<0.001, **p<0.005, ***p<0.01, ****p<0.05 in a Student's t test. e. NRP-1 mediates EC survival in zebrafish; the C-terminal three amino acids (SEA-COOH) are essential for the function. Fluorescent image of embryos injected with indicated morpholinos and mRNA, then subjected to the antibody stain with anti-GFP antibody and TUNEL assay to detect apoptosis. Insets show a magnification of apoptotic foci in the vessel. The arrows indicate apoptotic EC. i. Control; ii. Mismatch MO (4.5 ng); iii. zNRP-1a/1b MOs (4.5 ng+4.5 ng); iv. zNRP-1a/1b MOs (4.5 ng+4.5 ng)+hNRP-1 mRNA (0.3ng); v. zNRP-1a/1b MOs (4.5ng+4.5ng)+hNRP-1&detla;SEA mRNA (0.3 ng). f. The dynamics of NRP-1-induced EC apoptosis in the zebrafish embryo. Fluorescent images of different stage embryos injected with indicated morpholinos and subjected to the antibody stain and TUNEL assay to detect apoptosis. Insets show a magnification of apoptotic foci in the vessel. The arrows indicate apoptotic EC. i. Control; ii. zNRP-1a/1b MOs (3.0 ng+3.0 ng).
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