Figure 4. SUN1 stabilizes endothelial cell-cell junctions and regulates junction integrity.

(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated knockdowns (KD) in monolayers. Endothelial cells were stained for DAPI (cyan, DNA) and VE-cadherin (white, junctions). Insets show junctions. Scale bar, 10 µm. (B) Representative graph of impedance measured by real time cell analysis (RTCA). (C) Quantification of % change in cell index for RTCA measured at 5 hr. Normalized to non-targeting (NT) cell index. n=5 replicates. *, p<0.05 by Student’s two-tailed unpaired t-test. (D) Representative images of HUVEC with indicated siRNAs plated on biotinylated fibronectin and exposed to 15 dyn/cm² shear stress for 72 hr then treated with streptavidin. Endothelial cells were stained for DAPI (cyan, DNA), streptavidin (green), and VE-cadherin (white, junctions). Arrow indicates flow direction. Insets show junctions. Scale bar, 20 µm. (E) Quantification of cell alignment shown in (D). n=59 cells (NT) and 73 cells (SUN1 KD) compiled from three replicates. (F) Quantification of streptavidin area shown in (D). n=15 ROIs (NT) and 15 ROIS (SUN1 KD) compiled from three replicates. (G) Representative images of HUVEC with indicated siRNAs showing adherens following EDTA washout. Endothelial cells were stained for DAPI (cyan, DNA) and VE-cadherin (white, junctions). Insets show junctions. Scale bar, 20 µm. (H) Quantification of VE-cadherin line scans at 20, 40, and 60 min post EDTA washout in (G). 20 min: n=31 junctions (NT) and 23 junctions (SUN1 KD); 40 min: n=49 junctions (NT) and 33 junctions (SUN1 KD); 60 min: n=33 junctions (NT) and 33 junctions (SUN1 KD) compiled from three replicates. ns, not significant; ****, p<0.0001 by Student’s two-tailed unpaired t-test. For C, error bars represent standard deviation. For E, F, and H, boxes represent the upper quartile, lower quartile, and median; whiskers represent the minimum and maximum values.

Figure 4—figure supplement 1. SUN1 regulates endothelial junction integrity.

(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated siRNAs. Endothelial cells were stained for DAPI (cyan, DNA) and VE-cadherin (white, junctions). Insets show junctions. Scale bar, 20 µm. (B) Representative western blot showing levels of VE-cadherin protein in HUVEC with indicated siRNAs. GAPDH was used as a loading control. Quantification of VE-cadherin levels compiled from four replicates. ns, not significant by Student’s two-tailed t-test. (C) Representative images of HUVEC with indicated siRNAs cultured on biotinylated fibronectin and treated with streptavidin upon confluence. Endothelial cells were stained for DAPI (cyan, DNA), streptavidin (green), and VE-cadherin (white, junctions). Insets show junctions. Scale bar, 20 µm. (D) Quantification of streptavidin area shown in (C). n=29 ROIs (non-targeting [NT]) and 29 ROIs (SUN1 knockdown [KD]) compiled from four replicates. *, p<0.05 by Student’s two-tailed unpaired t-test. (E) Representative images and graphs of HUVEC with indicated siRNAs showing VE-cadherin line scan quantification. Endothelial cells were stained with DAPI (cyan, DNA) and VE-cadherin (white, junctions). Yellow line indicates where line scan was taken. Arrows point to the area under the curve (AUC). Scale bar, 20 µm. (F) Representative images of HUVEC with indicated siRNAs showing VE-cadherin staining after internalization assay. Endothelial cells were stained for DRAQ7 (cyan, DNA), VE-cadherin (white, junctions), and Phalloidin (green, actin). Scale bar, 20 µm. (G) Quantification of area of internalized VE-cadherin in (F). n=57 cells (NT) and 82 cells (SUN1 KD) compiled from three replicates. **, p<0.01 by Student’s two-tailed unpaired t-test. For B, error bars represent standard deviation. For D and G, boxes represent the upper quartile, lower quartile, and median; whiskers represent the minimum and maximum values.