Fig. 2: CHFR depletion in endothelial cells prevents VE-cadherin ubiquitylation, degradation, and endothelial barrier breakdown.

a HLMVEC were transfected scrambled-siRNA (Sc-siRNA) or CHFR-siRNA1. At 72 h post-transfection, cells were used for IB analysis (n = 2 independent experiments). b HLMVEC transfected with 100 nM of either Sc-siRNA or CHFR-siRNA1 were challenged with LPS (5 μg/ml) for the indicated times and cell lysates were used for IB. Shown are mean values ± SEM (n = 3 independent experiments; two-way ANOVA followed by Tukey’s post-hoc test). c HLMVEC transfected with Sc-siRNA or CHFR-siRNA1 as above were preincubated with or without proteasomal inhibitor MG132 (10 μM) for 2 h, and exposed to LPS. Cell lysates were immunoprecipitated (IP-ed) with anti-VE-cad pAb and blotted with antibodies specific to K⁴⁸-linked poly-Ub (K⁴⁸-Ub) or K⁶³-linked poly-Ub (K⁶³-Ub) (n = 2 independent experiments). d cells were stained with anti-VE-cad pAb. red, VE-cadherin (VE-cad); blue, DAPI. e, f cells were used to measure LPS- or PAR-1 activating peptide (TFLLRNPNDK-NH₂; 25 μM)-induced changes in TER as a measure of endothelial permeability changes. Arrow indicates time of LPS, PAR-1 peptide, or media addition. Shown are mean values ± SEM (n = 3 independent experiments; unpaired two-tailed Student’s t test). g Confluent HLMVEC exposed to LPS for indicated time intervals were stained with antibodies to VE-cadherin (red), CHFR (green), and DAPI (blue). Confocal images were used to assess co-localization of VE-cadherin with CHFR by Spearman’s rank correlation. Shown are mean values ± SEM (n = 10 field of view/group; one-way ANOVA followed by Tukey’s post-hoc test). Right panel shows magnified images. Arrows show co-localization of CHFR with VE-cadherin at AJs or in endocytosed vesicles. Red, VE-cadherin; Green, CHFR; Blue, DAPI; Scale bar = 10 μm.