Scribble knockdown prevents polarization of cells at the leading edge. (a) MCF10A monolayers scratch wounded, fixed 6 h following wounding and labelled with GM130 antibody to stain the Golgi (green) and TOPRO3 to label nuclei (blue). Shown beneath is a graphical representation of the position of the Golgi relative to the centre of the nucleus in cells at the leading edge. (b) Quantitation of cells at the leading edge with the Golgi polarized within a 120° arc behind the nucleus following: no wound (black) or 6 h following wounding (grey). Error bars represent s.d. of the mean. (c) Cells at the leading edge of a migrating monolayer stained with an α-tubulin antibody to label microtubule filaments. Staining is visible only in leading edge cells because these cells have a flattened morphology and the plane of the confocal sections is too basal to detect significant tubulin staining in other cells. (d) shEGFP and shScrib6 MCF10A cells were scratch wounded to remove approximately 50% of cells on the culture dish and levels of GTP-bound Cdc42 were measured by PAK-CRIB pulldown assay. Shown below is quantitation of Cdc42-GTP levels measured using LiCor Western blotting. Wounding caused only a slight increase in the level of total GTP-bound Cdc42 over the cell population in both control and ScribbleKD cells. GTPγS and GDP loaded controls are shown on the left.