Expression of mutant (m4) FL-APC leads to reduced F-actin levels at FAs and severe defects in MT-induced FA turnover. (A) Representative images of FA turnover cells after nocodazole treatment and washout at different time points. Cells from mock-treated (control) U2OS cells, cells depleted of endogenous APC (si-APC), and depleted cells rescued by plasmids expressing si-resistant FL-APC constructs (rescue FL-WT and FL-m4) were treated with nocodazole, and then the drug was washed out with serum-free media and cells were immunostained with phospho-Paxillin and tubulin antibodies. (B) Quantification of integrated fluorescence intensity of individual FAs measured from phospho-Paxillin immunostaining images as in A. Data were normalized to control cells treated with nocodazole before washout and averaged from three independent experiments (n ≥ 1,500 FAs per condition from 20 cells). Error bars, SEM; one-way ANOVA Holm-Sidak’s multiple comparison test. (C) Western blot analysis of phospho-Paxillin (P-Pax), total Paxillin, FAK-pY397 (P-FAK), and tubulin (Tub; loading control) levels in U2OS cells grown on plastic dishes, which is distinct from the cells in A grown on collagen-coated glass coverslips. This difference results in small differences in the timing of FA disassembly in A versus C. (D) Representative images of cells treated with nocodazole for 3 h showing merge of F-actin organization (rhodamine-phalloidin) and FAs (P-Paxillin). For each condition, representative magnifications of two boxed regions are shown. (E) Quantification of the ratio of fluorescence intensity of the F-actin signal to the phospho-Paxillin signal at individual FAs, measured from images as in D. Data were averaged from three independent experiments (>700 individual FAs and n = 50 cells per condition). Error bars, SEM; one-way ANOVA Holm-Sidak’s multiple comparison test. Statistical differences: ***, P < 0.001; ****, P < 0.0001; n.s., not significant.