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. 2018 Feb 26;9:819. doi: 10.1038/s41467-018-03186-z

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© The Author(s) 2018

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. 3 — EGFL7-KO is associated with an earlier onset of EAE and an increased CNS immune cell infiltration. a EGFL7 expression (red) using FISH and immunofluorescence (claudin 5, green) in cerebellum, brainstem, and spinal cord of WT C57Bl/6 naïve control versus MOG_35–55-immunized mice at EAE onset (d10), peak (d14) and remission (d25). Images (brainstem) representative of n ≥ 10 sections/mouse, 5 mice/time point. b, c Semi-quantitative analysis of EGFL7 expression according to (b) mean pixel intensity and (c) relative pixel intensity (mean pixel intensity × area). d EAE course and e proportion of asymptomatic mice in MOG_35–55-immunized EGFL7-KO versus WT; n = 12 mice/group, two pooled independent experiments. f Numbers of CD45^High immune cells in spleen and CNS (brain and spinal cord) and g cytokine expression by CNS-infiltrating CD4 T lymphocytes. n = 5–6 mice/group, EAE d11. h, i BBB integrity in brain and spinal cord of EGFL7-KO versus WT: h Western Blot for serum albumin n = 5 mice/group, EAE d11; i in vivo permeability assay with fluorescently-labeled dextrans. n = 8 mice/group, EAE d13. j–l IgG leakage assessed by (j) immunofluorescence on spinal cord sections from representative EGFL7-KO or WT mice. k, l Semi-quantitative analysis of IgG signal (green) in CNS (cerebellum, brainstem, spinal cord) according to (k) mean and (l) relative pixel intensities. n ≥ 5 sections/mouse, 4 mice/group, EAE d10. m–o Localization of CD4+ cells in cerebellum, brainstem, and spinal cord of EGFL7-KO versus WT, n = 5 mice/group, EAE d13. m Representative images (brainstem). CD4+ cells (purple) without contact to collagen IV (green) considered invading into CNS parenchyma (^); cells in the immediate vicinity of collagen IV considered perivascular (*). n Total number/mm² and o proportion of CD4+ cells invading CNS with respect to their localization. p EAE course and q proportion of asymptomatic mice in MOG_35–55-immunized Egfl7fl/fl;Cdh5(PAC)-CreERT2 males versus controls; n = 6 and 9 mice/group, respectively, two pooled independent experiments. Scale bars = 10 µm (a), 50 μm (j, m). Statistical analysis: one-way ANOVA with Tukey’s test (b, c), two-way repeated measures ANOVA with multiple comparisons test (d, p), Mantel-Cox test (e, q), Mann-Whitney U-test (b, c, f–o). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data presented as mean ± SEM