Figure 9.

Constitutively activated R-Ras fails to restore N-cadherin and Cx43 expression upon PGE2 treatment but restores cell adhesion of pericytes. (a) HBVPs transduced with mock or constitutively active R-Ras38V were treated with PGE2 (100 nM) or DMSO. N-cadherin and Cx43 expression was subsequently analyzed by western blot. (b) Mock- and R-Ras38V-transduced HBVPs (green) were treated with PGE2 for 72 h prior to coculture with HUVECs (red) on tissue culture plates. Cells were stained for N-cadherin (left panels) and Cx43 (right panels) 18 h later. 3D-surface plots of immunofluorescence intensity are used to quantify proteins at the cell–cell interface upon PGE2 treatment. One representative picture out of at least 10 regions measured for each condition is shown. The immunoreactivity is shown with arbitrary unit. Scale bar 50 μm. (c) Mock- and R-Ras38V-transduced HBVPs were incubated with PGE2 (100 nM) for 72 h, detached from the cell culture dish using 0.005% trypsin and seeded in 24-well plates for 30 min in basal medium at 37 ºC. Unattached cells were removed by PBS wash, and cells were fixed and stained with a methanol/crystal violet solution. Crystal violet was subsequently extracted from attached cells to measure the absorbance at 595 nm. One-way ANOVA test, *p < 0.05; ns: not significant.