Figure 1.
Co-culture of endothelial monolayers with A2058 melanoma cells induces endothelial cell-cell junction disruption local to melanoma cell adhesion sites.
(A) Phase contrast images of HUVEC monolayers with corresponding immunofluorescence staining of VE-cadherin junctions. VE-cadherin staining indicates existence of cell-cell junction integrity in the endothelial monolayer. With A2058 co-culture, VE-cadherin staining shows dissolution of cell-cell junctions. White arrows indicate the location of A2058 melanoma cells. (B) Quantification of the percentage gap formation between endothelial cells with A2058 melanoma cell co-culture with and without inhibitors of cellular contractility, actin remodeling, and Src kinase signaling. *p<0.001 in comparison to HUVEC only controls. #p<0.001 in comparison to HUVEC/A2058 co-culture sample incubated with DMSO. Scale bars, 25 µm.
Figure 2.
Schematic of network model connections.
Pathways are distinguished by color with IL-8 in blue, VCAM-1 in orange, IL-1β in green, c-Src in magenta, and model outputs in black. Grey indicates locations where all pathways converge. Phosphorylated proteins are indicated with the letter P and protein activation is designated with an asterisk.
Figure 3.
Computational simulations show that melanoma signals differentially affect endothelial contractility, actin remodeling, and junction disassembly.
Phosphorylation dynamics as a function of time for (A) MLC, (B) HSP-27, and (C) VE-cadherin following activation of VCAM-1, IL-8, and IL-1β individually or simultaneous activation of all inputs together.
Figure 4.
Simulations reveal that knockout of p38 MAPK influences MLC and VE-cadherin phosphorylation dynamics and completely inhibits HSP-27 phosphorylation.
Phosphorylation dynamics as a function of time for p38 on and knockout of p38 for (A) MLC, (B) HSP-27, and (C) VE-cadherin.
Figure 5.
Simulations show that knockout of MLCK reduces MLC phosphorylation, whereas HSP-27 and VE-cadherin phosphorylation is minimally affected.
Phosphorylation dynamics as a function of time for MLCK on and knockout of MLCK for (A) MLC, (B) HSP-27, and (C) VE-cadherin.
Figure 6.
Knockout of PKC affects the simulated dynamics of MLC and VE-cadherin phosphorylation.
Phosphorylation dynamics as a function of time for MLCK on and knockout of MLCK for (A) MLC, (B) HSP-27, and (C) VE-cadherin.