LIMK activity is required for collective invasion. (A) Double LIMK1/2 knockdown (siLIMK1&2) in MDA-MB-231 breast cancer cells cultured on glass coverslips dramatically reduced cytoskeletal F-actin staining. Microscope settings were kept constant between conditions to assess staining intensity differences. Bar, 40 µm. (B) X-Z stack view of MDA-MB-231 invasion into 3D matrix revealed inhibition by LIMK1/2 knockdown. Bar, 25 µm. (C) Confocal images taken at 15-µM intervals through the 3D matrix revealed significant inhibition of invasion for two independent siRNA duplex pairs against LIMK1/2 relative to NT control siRNA. Individual targeting of LIMK1 (siL1) or LIMK2 (siL2) did not significantly reduce invasion. Bar, 100 µm (average ± SEM, n = 3). (D) Recombinant TAT-Cofilin protein severed F-actin in vitro. (E) Recombinant TAT-Cofilin reduced phalloidin-stained F-actin structures in MDA-MB-231 breast cancer cells cultured on glass coverslips. Confocal microscope settings were kept constant between conditions. Bar, 40 µm. (F) Addition of cell-permeable TAT-Cofilin significantly inhibited 3D matrix invasion relative to equal mass of TAT-GST control. Bar, 100 µm (average ± SEM, n = 3).