APC/β-catenin-rich complexes at protrusion ends are dependent on the microtubule cytoskeleton. (A) MDCK cells were treated with 10 μg/ml nocodazole, or DMSO, in combination with 15 μM ALLN and stained with anti-β-catenin (green) and -tubulin (red) antibodies. Microtubule depolymerization inhibited protrusion formation by ALLN in nocodazole (Noc) post- and pre-treated cells compared to controls (No Noc). 630X. (B) Quantification shows that protrusions are inhibited by nocodazole treatment, regardless of whether nocodazole and ALLN were added simultaneously (ALLN+Noc), or ALLN was added first (ALLN/Noc) or second (Noc/ALLN) (*p < 0.05). (C) 4T07 cells were treated with 10 μg/ml nocodazole or DMSO (control) for 4, 9 or 12 h and stained with antibodies against β-catenin (green) and APC (red). Arrows illustrate punctate APC and β-catenin localization at protrusion ends in control cells. Nocodazole-treated cells lack this localization but demonstrate β-catenin at cell-cell contacts (arrows) that increases as the morphology becomes more epithelial-like over the time course. 630X. (D) 4T07 cells were transiently transfected with scrambled (control) or APC-specific siRNAs and stained with anti-β-catenin (red) and –APC antibodies (green). Control cells demonstrate APC and β-catenin co-localization at protrusion ends (arrows), while APC siRNA-transfected cells have a more rounded morphology and increased β-catenin localization at cell-cell contacts (arrows). Images were taken with the same exposure level for APC; cytosolic staining is non-specific. 630x.