Figure 3

Depletion of RhoB inhibits in vitro vessel sprouting and capillary morphogenesis. (A) RhoB-depleted HUVEC have reduced ability to form vessel sprouts on collagen I gels. Cells were either mock transfected with PBS or transfected with 20 nM siRNAs (Control, RhoB siRNA 1, RhoB siRNA 2) as indicated and seeded onto collagen I gels at 24 h post transfection. At time zero (48 h post-siRNA transfection), cells were placed in EGM-2 growth media supplemented with 50 ng/ml VEGF to induce sprouting. Media supplemented with VEGF was refreshed every two days. Blinded counts were made from 10 random fields of view from each of duplicate plates, and compiled data are presented as mean ± SE of duplicate dishes from each of two independently performed experiments. Significance was determined by unpaired t-tests relative to control siRNA at each respective time point (* represents p < 0.05 and ** represents p < 0.0001) (B) Depletion of RhoB levels inhibit capillary morphogenesis. HUVEC were transfected with siRNAs as indicated and seeded onto polymerized basement membrane extract (BME) in EGM-2 growth media. Images were taken 24 h after cell seeding and capillary morphogenesis was determined by averaging the total number of cord-like structures in 4 fields of view as assessed with ImageJ software. The data is presented as the mean ± SE of two independently performed experiments. Significance was determined by ANOVA with Fisher's PLSD post hoc analysis (* represents p < 0.05 and ** represents p < 0.0001).