LIMK1 regulates vesicular transport of MT1-MMP. A) Immunolocalization of MT1-MMP and LIMK1 showed strong staining of both proteins in the Golgi areas (White staining in the DIC merge and long white arrows) along with Golgi marker: TGN46. Yellow arrows: Colocalization of MT1-MMP and LIMK1 in Golgi vesicles at various distance towards plasma membrane: White arrows: Colocalization of MT1-MMP and LIMK1 to the plasma membrane. B) Analysis of Pearson's correlation coefficient of colocalization between LIMK1 and TGN46, MT1-MMP and TGN46, and LIMK1 and MT1-MMP in Golgi vesicles and in the membrane areas (insert in A: yellow arrows), C) Loss of expression and plasma membrane localization of MT1-MMP in PC3 cells following knock down of LIMK1 expression. Immunofluorescence analysis of MT1-MMP, and TGN46 expression and localization in PC3 cells transfected with scrambled RNA (green) (upper panel) or LIMK1 shRNA (green)(lower panel) expressing vectors. Upper panel: Yellow arrows: Colocalization of MT1-MMP with TGN46 in Golgi vesicles moving towards the plasma membrane, White arrow: Targeting of MT1-MMP to the plasma membrane. Lower panel: Colocalization of MT1-MMP and TGN46 to the perinuclear region (yellow arrow) but not in the transport vesicles (orange arrow). White arrow: Transport of TGN46 positive Golgi vesicles to the plasma membrane. No targeting of MT1-MMP could be noted in these cells. A significant reduction in the MT1-MMP concentration was also evident in these cells. Scale: 10 μm. D) Quantitative analysis of the staining intensity of MT1-MMP in the Golgi vesicles (red circles in DIC merge: yellow arrow) and in the membrane (entire membrane) in scrambled RNA and LIMK1 shRNA transfected PC3 cells. Pearson's correlation coefficient analysis confirms colocalization between MT1-MMP and TGN46 in Golgi vesicles at the same region used for the analysis of staining intensity. E and F) Immunofluorescence and quantitative analyses of the reduction of LIMK1 expression in LIMK1 shRNA but not scrambled RNA expression vector transfected cells. G) Western blots of MT1-MMP in the lysates of PC3 cells expressing scrambled shRNA or LIMK1 shRNA used for immunolocalization assays.