Fig. 3: FLRT2 controls VE-cadherin recycling at EC junctions.
From: Vascular FLRT2 regulates venous-mediated angiogenic expansion and CNS barriergenesis
a Proximity ligation assay (PLA) in HUVEC. Cells were stained with phalloidin (actin filaments) and DAPI (nuclei). b PLA signal quantification as puncta per cell number (DAPI) in the field. n = 20 and 23 pictures per condition from 3 experiments. Two-tailed Mann-Whitney test, p < 0.0001. c Immunoprecipitation (IP) of VE-cadherin and immunodetection of FLRT2 and VE-cadherin from mouse brain lysates. TL, total lysate. d FLRT2 immunoblot from HUVECs treated with Flrt2 and control siRNA. Loading control: β-actin. e FLRT2 protein quantification in control and Flrt2-siRNA treated HUVECs. n = 6 independent experiments. Two-tailed unpaired t-test, p = 0.001. f Antibody feeding assay in HUVEC transfected with control and Flrt2-siRNA treated with chloroquine. Cells immunostained for internalized VE-cadherin, total VE-cadherin and DAPI. Intensity of internalized VE-cadherin shown in arbitrary units (AU, upper panels). g Fluorescence intensity quantification of internalized VE-cadherin per cell. n = 84 control, 115 Flrt2-siRNA cells from 3 experiments. Two-tailed Mann-Whitney test, p < 0.0001. h Quantification of VE-cadherin intensity per cell-junction length. n = 22 control, 18 Flrt2 siRNA-treated pictures from 3 experiments. Two-tailed unpaired t-test, p = 0.029. i VE-cadherin and α-tubulin (loading control) immunoblots in HUVEC transfected with control- and Flrt2-siRNAs. j Quantification of VE-cadherin/loading control and cleaved VE-cadherin/loading control ratios. n = 11 (total), 8 (cleaved) experiments. Two-tailed unpaired t-test, p = 0.074 (total), p = 0.0001 (cleaved). k Primary mouse brain ECs (pmBEC) from control and Flrt2iΔEC littermates stained for Calpain-2 and DAPI. l Calpain-2 fluorescence intensity quantification per cell. n = 28 and 51 cells per condition, from 1 control and 1 Flrt2iΔEC littermate. Two-tailed Mann-Whitney test, p = 0.011. m Vessels stained for Calpain-2 and IB4 from control and Flrt2iΔEC littermates. n Calpain-2 fluorescence intensity quantification in vessels. n = 5 controls, 4 mutants. Two-tailed unpaired t-test, p = 0.002. Scale bars: 40 μm (a, upper panels), 15 μm (a, lower panels), 10 μm (f, k), 5 μm (m). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant.
