Inhibition of invadopodia formation reduces levels of active secreted MMP9 and matrix degradation. (A) Focalized MMP9 immunostaining in MDA-MB-231 cells plated on gelatin was reduced after LIMK1/2 knockdown. Bar, 10 µM. (B) RFP-labeled MDA-MB-231 cells plated in matrix-containing dye-quenched (DQ) collagen reveal considerable proteolytic activity that was reduced by LIMK1/2 knockdown. Bar, 20 µm. (C) LIMK1/2 knockdown in MDA-MB-231 cells plated on plastic, which decreased cofilin phosphorylation, did not alter MT1-MMP or MMP9 protein levels in cell lysates but did reduce levels of MMP9 activity in conditioned media. (D) Although LIMK1/2 knockdown in MDA-MB-231 cells plated on plastic reduced soluble MMP9-like activity, there was no effect on MMP9 expression as determined by RT-PCR. (E) Inhibition of invadopodia formation on gelatin by stable Fascin knockdown was associated with reduced levels of MMP9 activity in conditioned media but not in lysates from cells plated on plastic. Microscopy settings were varied to acquire images of similar intensities. Bar, 10 µm (averages ± SD, n = 180). (F) Knockdown of MMP9 significantly inhibited invasion into 3D matrix relative to control NT siRNA. Western blot shows MMP9 knockdown compared with AKT loading controls. Bar, 200 µm (averages ± SEM, n = 3).