Figure 3
Figure 3. PKCζ and β-catenin are required for Ang-1–induced persistent cell migration and for leading edge formation. (A) Quantification of the percentage of single cells after 6 hours of migration in PKCζ down-regulated cells in response to Ang-1 or VEGF stimulation (6 hours). Data are represented as mean ± SEM (*P < .05 compared with nonstimulated cells; †P < .05 compared with CT-siRNA–transfected cells). (B) Quantification of the persistence of cell migration in PKCζ down-regulated cells in response to Ang-1 or VEGF stimulation (6 hours). Data are represented as mean ± SEM (*P < .05 compared with nonstimulated cells; †P < .05 compared with Ang-1–stimulated CT-siRNA–transfected cells). (C) Representative vector diagrams (250 μm) of cell trajectories (top) and directionality of migration displayed in rose plot diagrams (bottom) of CT-siRNA and PKCζ-siRNA–transfected cell in response to Ang-1 or VEGF stimulation (6 hours). (D) Representative images taken from the wound edge at the initiation of imaging (0 hours) and 6 hours after wounding of BAEC monolayers. The migration front was traced to delimit the displacement of cells, and cells that detached from the edge were outlined. (E) Quantification of the percentage of single cells after 6 hours of migration and (F) persistence of cell migration in β-catenin down-regulated cells in response to Ang-1 stimulation. Data are represented as mean ± SEM (*P < .05 compared with nonstimulated cells; †P < .05 compared with Ang-1–stimulated CT-siRNA–transfected cells). (G) Representative confocal micrographs of immunofluorescence staining of PKCζ (red) and β-catenin (green) in BAECs located at the leading edge of a wound during migration in presence of Ang-1 or VEGF (30 minutes). Note that in contrast to control and VEGF-stimulated cells, Ang-1 induces the colocalization of PKCζ and β-catenin (merged in yellow) at the leading front of cells. Scale bar represents 20 μm. (H) Immunofluorescence staining showing that transfection of BAECs with PKCζ-siRNA, β-catenin-siRNA or treatment with the pseudosubstrate inhibitors (Myr-PS-PKCζ) prevents the colocalization of PKCζ (red) or β-catenin (green) at the leading edge of migrating cells after Ang-1 stimulation (30 minutes). Arrowheads point to β-catenin staining at cell-cell junctions. The leading edge of cells transfected with PKCζ-siRNA or β-catenin-siRNA is outlined to delimit the migration front. Scale bar represents 20 μm.

PKCζ and β-catenin are required for Ang-1–induced persistent cell migration and for leading edge formation. (A) Quantification of the percentage of single cells after 6 hours of migration in PKCζ down-regulated cells in response to Ang-1 or VEGF stimulation (6 hours). Data are represented as mean ± SEM (*P < .05 compared with nonstimulated cells; †P < .05 compared with CT-siRNA–transfected cells). (B) Quantification of the persistence of cell migration in PKCζ down-regulated cells in response to Ang-1 or VEGF stimulation (6 hours). Data are represented as mean ± SEM (*P < .05 compared with nonstimulated cells; †P < .05 compared with Ang-1–stimulated CT-siRNA–transfected cells). (C) Representative vector diagrams (250 μm) of cell trajectories (top) and directionality of migration displayed in rose plot diagrams (bottom) of CT-siRNA and PKCζ-siRNA–transfected cell in response to Ang-1 or VEGF stimulation (6 hours). (D) Representative images taken from the wound edge at the initiation of imaging (0 hours) and 6 hours after wounding of BAEC monolayers. The migration front was traced to delimit the displacement of cells, and cells that detached from the edge were outlined. (E) Quantification of the percentage of single cells after 6 hours of migration and (F) persistence of cell migration in β-catenin down-regulated cells in response to Ang-1 stimulation. Data are represented as mean ± SEM (*P < .05 compared with nonstimulated cells; †P < .05 compared with Ang-1–stimulated CT-siRNA–transfected cells). (G) Representative confocal micrographs of immunofluorescence staining of PKCζ (red) and β-catenin (green) in BAECs located at the leading edge of a wound during migration in presence of Ang-1 or VEGF (30 minutes). Note that in contrast to control and VEGF-stimulated cells, Ang-1 induces the colocalization of PKCζ and β-catenin (merged in yellow) at the leading front of cells. Scale bar represents 20 μm. (H) Immunofluorescence staining showing that transfection of BAECs with PKCζ-siRNA, β-catenin-siRNA or treatment with the pseudosubstrate inhibitors (Myr-PS-PKCζ) prevents the colocalization of PKCζ (red) or β-catenin (green) at the leading edge of migrating cells after Ang-1 stimulation (30 minutes). Arrowheads point to β-catenin staining at cell-cell junctions. The leading edge of cells transfected with PKCζ-siRNA or β-catenin-siRNA is outlined to delimit the migration front. Scale bar represents 20 μm.

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