Figure 2

siRNA-mediated RhoB depletion inhibits endothelial cell migration but not viability. (A) Efficient depletion of RhoB protein levels was achieved in HUVEC with small interfering RNA treatment. HUVEC were either mock transfected with PBS (M) or transfected with 20 nM of the indicated siRNAs [Control (C), RhoB siRNA 1, RhoB siRNA 2]. Cellular total protein lysates were collected at 48 h post transfection and analyzed for levels of RhoB by western blot. Levels of β-actin served as a protein loading control. (B) Assessment of cell viability in RhoB-depleted HUVEC. Cells were either mock transfected with PBS or transfected with siRNAs, as indicated, and number of viable cells were assessed following trypan blue exclusion and counting with a Beckman Coulter Vi-Cell XR analyzer in triplicate wells at each time point. Data presented shows mean ± SD of two independently performed experiments. (C) RhoB-depletion inhibits cell migration. A scratch wound assay of transfected HUVEC was performed in MCDB 131, with 0.05% FBS and 50 ng/ml VEGF. Images depict wound size at time of wounding (Time = 0 h) and 24 h later. (D) Analysis of cell migration from (C). Bars represent the mean ± SD for percent wound closure, after 24 h for a representative experiment. P values were calculated using a two-tailed unpaired student's t test (* P < 0.05).