Selective LIMK inhibitor affects actin dynamics and blocks collective invasion. (A) Structure of LIMK inhibitor (LIMKi). (B) LIMKi reduced cofilin phosphorylation in MDA-MB-231 breast cancer cells cultured on plastic in a dose-dependent manner as detected by Licor Odyssey two-color infrared scanning. (C) Cytoskeletal F-actin staining was reduced in MDA-MB-231 breast cancer cells cultured on glass coverslips by LIMK inhibitor. Confocal microscope settings were kept constant between the top three rows of images. Settings were varied in the actin high resolution (Res.) row to allow for comparable image acquisition of F-actin structures. Bar, 20 µm (top three rows) or 40 µm. (D) Actin fiber analysis by ArrayScan HCS Morphology Explorer BioApplication of MDA-MB-231 breast cancer cells grown on glass-bottomed dishes and treated with indicated LIMKi concentrations revealed no detectable changes in F-actin morphology but decreased overall staining intensity (averages ± SEM, n = 3). (E) Activation of the serum response factor transcriptional reporter by serum stimulation of MDA-MB-231 breast cancer cells cultured on plastic was comparably blocked by double LIMK1/2 knockdown or by LIMK inhibitor (averages ± SEM; left graph, n = 3; right graph, n = 5). (F) The cell index, measured by electrical impedance using the xCELLigence system, was measured over time for cells plated on fibronectin-coated wells in the presence of DMSO or increasing concentrations of LIMKi as indicated (averages of n = 3). (G) Confocal sectioning of 3D matrix invasion by MDA-MB-231 cells revealed a significant dose-dependent inhibition by LIMKi. Bar, 200 µm average ± SEM, n = 3).