Activation of RhoA restores cell shape and migration in Girdin knockdown cells. Confocal immunofluorescence (top) and reflectance (bottom) images showing actin filament (red) organization of (a) control; (b) Girdin knockdown, and (c) RhoA Activator treated Girdin knockdown MDA-MB-231 cells during 3D microtrack migration, Inset magnification 2X, White arrows indicate presence of actin stress fibers (red) in control and RhoA activator treated Girdin knockdown cells; (d) Average speed of control siRNA, RhoA activated control siRNA, Girdin siRNA and RhoA activated Girdin siRNA-treated cells during migration through microtrack; (e) average aspect ratio and (f) average circularity of control and RhoA treated Girdin knockdown cells; # of cells per treatment = 44–45, *p value < 0.05, ‘n.s.’ **p value > 0.05, Scale bars = 25 µm.