FIGURE 1.

Effects of siRNA-mediated MARCKS knockdown on endothelial cell wound healing. Shown are the results of a wound healing assay analyzed in cultured endothelial cells transfected 48 h previously with either control or MARCKS duplex siRNA constructs. A, selected time-lapse photomicrographs (magnification ×10) obtained at the indicated times after producing a linear wound in the endothelial monolayer; the borders of the wound at t = 0 are noted. Differential interference contrast images were recorded every minute for 8 h using live-cell imaging conditions in an Olympus DSU microscope; the experiment shown was repeated three times with equivalent results. B, pooled data from three independent experiments in which the percentage of the original wound that is occupied by cells is plotted as a function of time, and then quantitated using Image J software. The difference in the rate of wound recovery is highly significant between the control and MARCKS siRNA-transfected endothelial cells (p < 0.001, n = 3). C, the results of wound healing assays analyzed after transfection of endothelial cells with control or MARCKS siRNA. They were then studied following cell wounding in the presence of the vehicles, VEGF (20 ng/ml), and insulin (100 nm), as shown. The fractional repopulation of the wound by endothelial cells is shown on the ordinate, as in B. D, an immunoblot of endothelial cells transfected with control or MARCKS siRNA-targeting constructs, and probed with antibodies as shown.