Fig. 7: FLRT2 deficiency disrupts tight junctions and blood-brain barrier.
From: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis
a Immunoblot of cytosolic and nuclear fractions obtained from bEnd.3 cells treated with control and Flrt2-siRNA showing protein levels of β-catenin and FoxO1, and α-tubulin (cytosolic) and Lamin A/C (nuclear) as controls. β-catenin (b) and FoxO1 (c) protein levels relative to loading controls. n = 3 independent experiments. Two-tailed unpaired t-tests, p = 0.342 (cytoplasm), p = 0.016 (nucleus) (b); p = 0.126 (cytoplasm), p = 0.020 (nucleus) (c). d Immunoblot showing FLRT2 and Claudin-5, and β-actin (loading control) in control and Flrt2 siRNA-treated bEnd.3 cells. e Quantification of FLRT2 and Claudin-5 protein levels. n = 5 (FLRT2) and 6 (Claudin-5) independent experiments. Two-tailed unpaired t-test, p < 0.0001 and p = 0.026. f Immunoblot showing Claudin-5, and α-tubulin as loading control in total brain lysates from control and Flrt2iΔEC littermates. g Quantification of Claudin-5 protein levels. n = 8 animals per genotype. Two-tailed Mann-Whitney test, p value = 0.034. h Neocortical blood vessels stained for Claudin-5 and VE-cadherin. i Quantification of the ratio of split Claudin-5 junction length to the total junctional length. n = 5 control and 7 mutant mice. Two-tailed unpaired t-test, p = 0.006. j Representative fluorescent whole-brain images of control and Flrt2iΔEC littermates injected with AlexaFluorTM 555-conjugated cadaverine at P7-P8. k Cadaverine whole-brain intensity quantification in control and Flrt2iΔEC littermates. n = 16 control, 18 mutant animals. Two-tailed unpaired t-test, p = 0.018. l Brain cortices from cadaverine-injected control and Flrt2iΔEC littermates immunostained for Collagen IV (Col IV). m TEM representative images of EC junctions in brain capillaries in control and Flrt2iΔEC mice. Note the abnormal junctions often associated with the presence of vacuoles (red arrows) in Flrt2iΔEC vessels. n Incidence of abnormal junctions (left) and junctions with vacuoles (right) in control and Flrt2iΔEC brain capillaries. n = 3 animals per genotype. Two-tailed unpaired t-test, p = 0.001 (junctions), p = 0.021 (vacuoles). Scale bars: 10 μm (h), 1 mm (j), 100 μm (l), 200 nm (m). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P > 0.001.
