Fig. 7: Lymphatic secretion of reelin requires VE-cadherin plasma membrane junctional organization in vitro and VE-cadherin expression in vivo.
A Time course of reelin levels (ng/mL) in conditioned media collected from LECs treated with +/− 100 nM AM and quantified by ELISA. n = 3-8 biological replicates (mean ± s.d). Significance was determined using a Mixed-effects model (REML) with Šídák’s multiple comparisons test. To examine the effect of VE-cadherin membrane levels on reelin levels (ng/mL) in LEC conditioned media, LECs were treated with +/− 100 nM AM as well as pretreated with (B) shCDH5 (48 h), (C) 10 µM Rasarfin (4 h), (D) 100 µM Endosidin-2 (24 h), or (E) shEXOC5 (48 h) and quantified by ELISA. n = 3-8 biological replicates for each condition (mean ± s.d). Significance was determined using a Mixed-effects model (REML) (panel B), a two-way ANOVA with Geisser-Greenhouse correction (C and E), or a one-way ANOVA (panel D) with Tukey’s multiple comparisons test. Statistics only shown for comparisons between samples within a given time point. All comparisons are shown in Supplementary Fig. 10. Shown are representative western blots of plasma membrane fractions from LECs to correlate time-dependent secretion of reelin with changes in plasma membrane levels of VE-cadherin. Actin served as a load control. F Serum reelin levels of Cdh5ΔPROX1 and littermate Cdh5fl/fl (WT) mice before tamoxifen treatment at 1–2 months old (D1, hollow symbols) and 3 weeks after tamoxifen treatment (D22, solid symbols). Significance was determined using a two-way ANOVA with Šídák’s multiple comparisons test. n = 8 mice per group (mean ± s.d). F Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license. Precise n numbers and p-values are found for each condition in Supplementary Data 10. Source data are provided with this paper.*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
