LIMK activity is not required for 2D cell motility. (A) A modified scratch-wound assay in which stoppers were used to produce regular and reproducible cell-free zones was used to monitor cell motility. Diameter of circle = 2 mm. LIMK1/2 double knockdown with two independent siRNA duplex pairs did not affect MDA-MB-231 wound closing on uncoated glass-bottomed multi-well plates relative to NT control siRNA. Treatment with increasing LIMKi doses also did not affect wound closing (average ± SEM, n = 4). (B) The random migration of MDA-MB-231 cells transfected with NT or LIMK1/2 siRNA on an oriented 3D fibrillar ECM generated by primary cultured human dermal fibroblasts was determined by time-lapse microscopy. Tracks of individual cells revealed no obvious differences in path length, direction, or velocity. Bar, 100 µm. (C) Analysis of vectorial distance, accumulated distance, and persistence with ImageJ revealed no differences in 2D cell motility on fibroblast-derived matrix between NT or LIMK1/2 siRNA transfectants (average ± SEM, n = 4).