Figure 3

RKIP-mediated inhibition of cell invasion depends on the E-cadherin (E-cad) cell–cell junction localization. (a) (Top left panel) Representative western blots of lysates prepared from control knockdown (shGFP), or four different E-cad knockdowns (shEcad#2–5) 4T1 cells. (Bottom left panel) Representative results of invasion assays with same set of cells showing number of invaded cells through Matrigel in each indicated 4T1 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown in the bottom left panel. (b) (Top left panel) Representative western blots of lysates prepared from control knockdown (shGFP), or E-cad knockdowns BT20 cells. (Bottom left panel) Representative results of invasion assays with shGFP control knockdown or titrated E-cad knockdown showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown in the bottom left panel. (c) (Top left panel) Representative western blots of lysates prepared from BT20 cells with different combinations of controls, E-cad knockdown (shEcad) and RKIP expression as indicated. (Bottom left panel) Representative results of invasion assays with same set of cells showing number of invaded cells through Matrigel in each indicated BT20 cell lines (mean ± SE). (Right panel) Representative images of the stained invaded cells in indicated cell lines shown at the bottom left panel. (d) (Left panel) Representative western blots of lysates prepared from BT20 cells with different combinations of controls, RKIP knockdown (shRKIP¹⁷⁵) and RhoAQL expression as indicated. (Left, bottom) Representative images of immunofluorescent staining with β-Catenin Ab and DRAQ5 (Nucleus) of BT20 cells with control knockdown (shLUC), RKIP knockdown (shRKIP), empty vector control (EVC) or RKIP expression. (right panel) β-Catenin intensity quantification on cell–cell junctions of immunofluorescent images shown in the left panel using MetaMorph analysis software. Representative images of immunofluorescent staining with p120 Ab and DRAQ5 (Nucleus) of BT20 cells with control knockdown (shLUC), RKIP knockdown (shRKIP), empty vector control (EVC) or RKIP expression. (Right panel) p120 intensity quantification on cell–cell junctions of immunofluorescent images shown in the left panel using MetaMorph analysis software. Unpaired Student's t-test (two-tailed) was used for all analyses with p < 0.05 considered significant. ns not significant.