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Figure 4 | Vascular Cell

Figure 4

From: RhoB controls endothelial cell morphogenesis in part via negative regulation of RhoA

Figure 4

Depletion of RhoB modulates the activity of RhoC and RhoA, and the resulting increased RhoA activity contributes to the observed reduction in capillary morphogenesis. (A) G-LISA analysis indicating increased levels of activated RhoA in response to VEGF in cells with depleted RhoB expression. Cells were transfected with the indicated siRNAs, and 48 h post transfection were serum starved for 5 h in MCDB 131, and subsequently stimulated with 10 ng/ml VEGF in MCDB 131. At various times post-stimulation, protein lysates were generated and activated RhoA was detected by G-LISA assays as described in Materials and Methods. Bars represent the mean ± SE of a representative experiment performed in triplicate (* represents p < 0.05 as determined by unpaired t-tests when compared to Control siRNA samples at each time point). Similar trends were observed in independent experiments. (B) G-LISA analysis indicating reduced RhoC activity in cells treated with RhoB siRNAs. Cells were transfected with the indicated siRNAs (Control, RhoB siRNA 1, RhoB siRNA 2), followed by starvation overnight in MCDB 131 with reduced serum (0.5%), and for an additional 4 h in MCDB 131 (0% serum). Cells were subsequently stimulated with 50 ng/ml VEGF in MCDB 131 for 5 min and protein lysates were generated for analysis of RhoC activity (at 48 h post-siRNA transfection) by G-LISA as described in Materials and Methods. Bars indicate the mean ± SE for duplicate measures in each of two independently performed experiments. (C) Protein levels of RhoA and RhoC were assessed in RhoB siRNA-treated HUVEC. Cells were transfected with 20 nM of the indicated siRNAs and lysates collected at 48 h post transfection. Protein levels were analyzed by western blotting, with levels of β-actin serving as a control for protein loading. (D) Defective capillary morphogenesis in RhoB depleted HUVEC is restored upon inhibition of RhoA activity. HUVEC were transfected with siRNAs as indicated and seeded onto BME in EGM-2 growth media in the absence or presence of 0.25 μg/ml C3 transferase or DMSO as a vehicle control. Images are representative photos taken 24 h after cell seeding. (E) Analysis of capillary morphogenesis from (D). Capillary morphogenesis was determined by averaging the total number of cord-like structures in 4 fields of view in triplicate wells as assessed with ImageJ software. Bars represent the mean ± SE of duplicate wells from each of two independently performed experiments (* represents p < 0.05 and ** represents p < 0.0001 compared to control siRNA as determined by ANOVA with Fisher's PLSD post hoc testing).

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