The tripeptide sequence GCI of RhoB is critical for cell motility. (A and B) Quantification of length (A) and half-life (B) of FAs was scored from transfected SKBr3 cells after HGF stimulation (20 min). CTL, control. (C) Fluorescence was bleached (arrowheads) within the stable FAs of control, Arf6 KD, RhoB KD, or RhoBΔGCI at the leading edge of migrating SKBr3 cells, and fluorescence recovery was monitored over time. (D) Quantification of Paxillin-EGFP fluorescence over time and percentage of recovery (box) are presented for Arf6 KD, RhoB KD, or RhoBΔGCI cells. (E) Phase-contrast images of HGF-treated MDA-MB-231 cells on collagen/Geltrex matrix transduced with Arf6 or RhoB shRNAs and rescued by RhoB WT or RhoBΔGCI. (F and G) Quantification of the speed (F) and distance from the origin (G) of MDA-MB-231 cells transduced with shRNAs and transfected with cDNAs as indicated, cultured on collagen/Geltrex matrix and stimulated with HGF. The distance from the origin was determined as the net displacement between the initial position and the final position observed during an 8-h period. All quantified data indicate mean values ± SEM from three independent experiments. Scale bar = 10 µm; 2 µm for magnification. P values based on comparisons with control: n.s., nonsignificant. *, P < 0.05; **, P < 0.001.