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. 2022 Jul 20;13:4188. doi: 10.1038/s41467-022-31904-1

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a NRP1 impairs VE-cadherin localization at endothelial cell-to-cell contacts. Confocal microscopy on control (siCtrl) or NRP1 (siNRP1) silenced ECs rescued with empty vector (Ctrl/siCtrl and Ctrl/siNRP1) or with mouse Nrp1 (Nrp1/siNRP1) and stained for NRP1 (magenta) and VE-cadherin (green) and quantification of VE-cadherin fluorescence intensity in AJs. Scale bar 10 µm. Mean ± SEM of three independent experiments (depicted in different colors). One-way ANOVA with Bonferroni correction; ***P value = 0.0004. b NRP1 silencing does not affect VE-cadherin protein levels. A representative experiment of three is shown. c FRAP analysis reveals NRP1 promotes VE-cadherin turnover at AJs in ECs transduced with VE-cadherin-mCherry and silenced or not for NRP1. Above, VE-cadherin fluorescence recovery = normalized signal in each cell before and after photobleaching to 100% and 0%. Three independent experiments (8 cells each) mean ± SEM is shown. Below, mobile fraction = fluorescence recovered post-bleach —after photobleaching/pre-bleach ROI fluorescence—after photobleaching. Mean of three independent experiments ± SEM is shown. Scale bar 5 µm. Two-way ANOVA with Bonferroni correction; ns = P value ≥ 0.05, *P value ≤ 0.05, **P value ≤ 0.01, ***P value = 0.000028. d NRP1 promotes VE-cadherin internalization. VE-cadherin endocytosis in Ctrl/siCtrl, Ctrl/siNRP1 or Nrp1/siNRP1 ECs measured by capture ELISA. Mean ± SEM of three independent experiments is shown. One-way ANOVA with Bonferroni correction; **P value = 0.0037, ***P value = 0.0003. e NRP1 sustains histamine-elicited endothelial permeability. Quantification of Dextran through Ctrl/siCtrl, Ctrl/siNRP1 or Nrp1/siNRP1 EC monolayer after histamine. Mean ± SEM of three independent experiments is shown. Two-way ANOVA with Bonferroni correction; *P value ≤ 0.05, **P value ≤ 0.01. f Vascular permeability reduction in Nrp1^EC−/− mice. Left, confocal microscopy on Nrp1^+/+ or Nrp1^EC−/− mice ear sections stained for NRP1 (magenta), MECA32 (green), and DAPI (blue). Scale bar 75 µm. Right, histamine permeability in Nrp1^+/+ or Nrp1^EC−/− mice (Evans Blue/skin weight). Data are mean ± SD (n = 5 per group). Two-tailed heteroscedastic Student’s t test; *P value = 0.0321. Source data are provided as Source Data file.