|
Fig. 5 SPTBN1 has a potential role in cell junction regulation, is associated with VE-cadherin at endothelial cell junctions and is linked to focal adhesion regulation. A Sptbn1 was silenced in HMEC-1, after which gene expression was measured by microarray analysis. GO analysis of the differentially expressed genes revealed links to regulation of adherens junctions and cell junctions, as well as focal adhesions. B Subsequent network prediction analysis showed clear involvement of sptbn1, but also confirmed its connection with dock1 and zeb1 in these pathways, and its link to junctional proteins like VE-cadherin (CDH5), focal adhesion proteins like paxillin (PXN), and cell–matrix related proteins like vinculin (VCL). C Immunofluorescent staining of HMEC1 with silenced sptbn1 (or siControl treated controls) for SPTBN1 (red) and the endothelial junction protein VE-Cadherin (green); nuclei were counterstained with Hoechst 33,342. As expected, silencing led to sharply reduced SPTBN1 expression, while interestingly also VE-Cadherin expression was reduced. The latter was seen to be more dispersed over the whole cell after sptbn1 silencing, but is enriched at stable, straight junctions between cells, as well as at the basolateral side. At junctions, SPTBN1 was seen to colocalize with VE-cadherin. D Immuno-precipitation analysis of HUVEC and HMEC-1 for SPTBN1, followed by immunoblotting for VE-cadherin and Ezrin (loading control) demonstrated direct interaction of SPTBN1 and VE-Cadherin for both cell types. TLC: Total cell lysate, IP is Immuno-precipitate (E) Knockdown of SPTBN1 led to an increase in stress fibers and an increased staining of p-paxillin at the basolateral side of the cell, indicative of an increase in the number of focal adhesions, and in all, an altered cell adhesion to the substrate (F + G) Quantitative image analysis revealed reduced junctional width after sptbn1 silencing (F), with a concomitant increase in paxillin and focal adhesions (p-paxillin) at the basolateral side of the cell (G). Data are representative images from three independent experiments (C–G); error bars indicate SEM (F, G); * p ≤ 0.05. Scalebars indicate 10µm. D, F, H, I); *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.005