Figure 10

Vascular endothelial (VE)-cadherin and zonula occludens (ZO)-1 in junctional actin organization. Human umbilical vein endothelial cells (HUVECs) were transfected with small interfering RNA (siRNA) oligonucleotides targeting VE-cadherin, ZO-1 or a non-specific oligonucleotide control. After 24 h cells were trypsinized and plated at confluence and analysed 48 h later (72 h after transfection). Twenty hours before the analysis cells were stimulated with tumour necrosis factor (TNF)-α in growth medium. (A) Cells were lysed and the effect of each siRNA on the levels of VE-cadherin, ZO-1 and transferrin receptor (TfR) were analysed by western blotting. (B, C) siRNA-transfected HUVECs were stimulated with TNF-α, fixed and stained for the indicated junctional proteins and actin filament (F-actin) (B) or F-actin and paxillin (C). (D) Quantitation of discontinuous junctions, F-actin content and focal adhesions in siRNA-treated cells upon TNF-α stimulation. * P < 0.045; ** P < 0.065.