Characterization of APC/β-catenin-rich clusters at membrane protrusion ends. (A) MDCK cells were treated for 24 h with 15 μM ALLN or DMSO, and phase-contrast microscopy demonstrated cell flattening and extensive membrane protrusions in drug-treated cells. 400X. (B) Total cell lysates were with anti-β-catenin and -β-actin antibodies. There was an increase in total β-catenin ALLN (range 0.5-30 μM); and the values shown represent densitometry of β-catenin normalized to actin relative to control cells. (C) MDCK cells were transfected with β-catenin or non-targeting siRNAs and treated with 15 μM ALLN or DMSO for 24 h, and stained with anti-β-catenin (red) or phalloidin (green). With β-catenin knockdown, there are very few protrusions with ALLN compared to adjacent areas in which the cells are β-cateninproficient (arrows). 630X. (D) MDCK cells were treated with 15 μM ALLN or DMSO (top) or 10 ng/ml TGFβ or ethanol for 24 h (bottom). The cells were stained with anti-β-catenin (red) and -APC (green) antibodies; arrows indicate co-localization of β-catenin and APC at membrane protrusions. 630X. (E) EpH4 and 67NR and 4T07 cells were stained with anti-β-catenin (red), –E-cadherin (green), and –APC (red) antibodies and phalloidin. Top, arrows indicate β-catenin localization to the lateral membrane in EpH4 and 67NR cells but to protrusions in 4T07 cells. Bottom, arrows indicate punctate APC localization at the basal surface in EpH4 and at membrane protrusions in 4T07 cells. 630X. (F) Hs587t human breast cancer cells show β-catenin (red) and APC (green) co-localization of membrane extension tips. 630X.