Figure 7. SUN1 regulates endothelial cell junctions through nesprin-1.

(A) Representative images of human umbilical vein endothelial cells (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. (B) Representative images of HUVEC with indicated siRNAs. Endothelial cells were stained for α-tubulin (cyan, microtubules) and VE-cadherin (red, junctions). Insets show α-tubulin contacts at junctions. Scale bar, 20 µm. (C) Representative images of HUVEC with indicated siRNAs. Endothelial cells were stained for DAPI (blue, DNA), α-tubulin (red, microtubules), and GEF-H1 (cyan). Insets show α-tubulin and GEF-H1 colocalization. Scale bar, 20 µm. (D) Quantification of streptavidin area shown in (A). n=22 ROIs (non-targeting [NT]), 22 ROIs (SUN1 knockdown [KD]), and 22 ROIs (SUN1/nesprin-1 KD) compiled from four replicates. ns, not significant; ***, p<0.001; ****, p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. (E) Quantification of peripheral α-tubulin area shown in (B). n=52 cells (NT), 52 cells (SUN1 KD), and 52 cells (SUN1/nesprin-1 KD) compiled from three replicates. ns, not significant; ***, p<0.001; ****, p<0.0001 by one-way ANOVA with Tukey’s multiple comparisons test. (F) Quantification of free GEF-H1 normalized to α-tubulin associated GEF-H1 shown in (C). n=32 cells (NT), 32 cells (SUN1 KD), and 32 cells (SUN1/nesprin-1 KD) compiled from three replicates. ns, not significant; **, p<0.01; ****, p<0.0001 by one- way ANOVA with Tukey’s multiple comparisons test. For all graphs, boxes represent the upper quartile, lower quartile, and median; whiskers represent the minimum and maximum values.

Figure 7—figure supplement 1. Loss of nesprin-1 alone does not impact endothelial junctions or microtubules.

(A) Representative images of human umbilical vein endothelial cells (HUVEC) with indicated siRNAs and indicated antibodies. Endothelial cells were stained for DAPI (DNA) and nesprin-1. Scale bar, 20 µm. (B) 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. (C) Quantification of streptavidin area shown in (B). n=24 ROIs (non-targeting [NT]) and 21 ROIs (nesprin-1 knockdown [KD]) compiled from three replicates. ns, not significant by Student’s two-tailed unpaired t-test. (D) Representative images of HUVEC with indicated siRNAs. Endothelial cells were stained for α-tubulin (cyan, microtubules) and VE-cadherin (red, junctions). Insets show α-tubulin contacts at junctions. Scale bar, 20 µm. (E) Quantification of peripheral α-tubulin area shown in (D). n=40 cells (NT) and 40 cells (nesprin-1 KD) compiled from two replicates. ns, not significant by Student’s two-tailed unpaired t-test. (F) Representative images of HUVEC with indicated siRNAs. Endothelial cells were stained for DAPI (blue, DNA), α-tubulin (red, microtubules), and GEF-H1 (cyan). Insets show α-tubulin and GEF-H1 colocalization. Scale bar, 20 µm. (G) Quantification of free GEF-H1 normalized to α-tubulin associated GEF-H1 shown in (F). n=20 cells (NT) and 20 cells (nesprin-1 KD) compiled from two replicates. ns, not significant by Student’s two-tailed unpaired t-test. For all graphs, boxes represent the upper quartile, lower quartile, and median; whiskers represent the minimum and maximum values.