Stimulation of β-catenin signals by HGF is dependent on endocytosis.

<p>(A) Western blot analyses of the cytosolic fraction of MDCK cells after treatment with HGF and increasing amounts of Dynasore (50, 100, 200 and 400 µM) for 6 and 16 hours shows a dose-dependent reduction in cytosolic β-catenin accumulation by HGF. (B) Western blot analysis of cytosolic fraction of MDCK cells after treatment with HGF and Dynasore (100 and 400 µM) for 30 minutes. Blocking endocytosis by Dynasore does not interfere with HGF signalling-induced phsophorylation of Erk (p-Erk). (C–H) Confocal microscopy of MDCK cells showing E-cadherin (green) and either β-catenin (red in C,E,G) or transferrin receptor (red in D,F,H) after treatment with HGF without or with Dynasore (400 µM) for 16 hours. In the absence of HGF or Dynasore (C,D), β-catenin and E-cadherin are localised at the cell membrane (C, also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023899#pone-0023899-g002" target="_blank">Fig. 2A,B</a>) while transferrin receptor is mostly in the subcortical area (D). HGF treatment (E,F) causes internalisation of β-catenin and E-cadherin (E, also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023899#pone-0023899-g002" target="_blank">Fig. 2E,F</a>) and movement of transferrin receptor to a perinuclear location, where it colocalises with internalised E-cadherin (F). In the presence of Dynasore, the ability of HGF to induce internalisation of β-catenin and E-cadherin is abolished, and they both remain at the cell membrane (G). Scale bars; 20 µm. (I) TOPflash reporter assay in MDCK cells with control (c) or β-catenin (β-cat) transfection and HGF treatment (similar to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023899#pone-0023899-g001" target="_blank">Fig. 1F</a>), in the absence or presence of Dynasore (400 µM). There is a reduction in Wnt pathway readout by both HGF and HGF plus β-catenin in the presence of Dynasore. HGF and Dynasore were added to transfected cells for the final 16 hours of incubation. Data were all normalised to the control without Dynasore. (J–L) Confocal microscopy of MDCK cells transfected with Flag-tagged β-catenin (red), treated with HGF and Dynasore as indicated, counterstained with DAPI (blue). Exogenous β-catenin is localised at the cell membrane in the absence of HGF (J), whereas HGF-treated cells show exogenous β-catenin in the cytoplasm, both in the absence (K) and presence (L) of Dynasore. Scale bar; 20 µm. (M) TOPflash reporter assay in MDCK cells with β-catenin transfection and HGF and Dynasore (200 µM) treatment. Where applicable, HGF were first added to cells for 24 hours to make cells mesenchymal, before adding Dynasore for 16 hours (total 40 hours of HGF). Dynasore attenuates the effect of transfected β-catenin in the mesenchymally transformed cells. *<i>P</i> = 0.011. (N) Schematic diagram summarising the requirement for the release of an active form of β-catenin into the cytosol.