Expression of mutant (m4) FL-APC causes severe defects in directed cell migration. (A) Representative wound-healing assays showing U2OS cells stained with SiR-actin, comparing mock-treated (control) cells, cells depleted of endogenous APC (si-APC), and depleted cells rescued by plasmids expressing si-resistant FL-APC (WT or m4). Cells are shown 0, 7.5, and 10 h after scratch. Yellow dashed lines mark wound edges. (B) Quantification of wound closure 10 h after scratch, as in A. Data averaged from six independent experiments performed in triplicate. Error bars, SD; one-way ANOVA Holm-Sidak’s multiple comparison test. (C) Quantification of mean wound size at t = 0. Data averaged from six independent experiments performed in triplicate. Error bars, SD; one-way ANOVA Holm-Sidak’s multiple comparison test. (D) Representative traces of the migration paths of individual U2OS cells (expressing GFP-actin) moving into a wound site, displayed in radial arrays, where the center of each array is the starting point in the migration path. Shown are 8 out of the 20 traces used for quantification in E. (E) Tortuosity index of the migration paths of individual cells from C, defined as the length of the path divided by the net distance migrated (L/D; n = 20 cells). Error bars, SEM; two-tailed Student’s t test. (F) Rose diagrams showing the migration angles of MDA-MB-231 cells in Dunn chamber chemotaxis assays, comparing mock-treated cells (control), cells depleted of endogenous APC (si-APC), and depleted cells rescued by plasmids expressing si-resistant FL-APC (WT or m4). Plots show data pooled from three independent experiments. (G) Y forward migration index for the same cells as in E. Error bars, SEM; two-tailed Student’s t test. Significant differences: *, P < 0.05; **, P < 0.01; ***, P < .001; ****, P < 0.0001; n.s., not significant.