Fig. 2

Implantation of two pancreatic cancer cell lines into the same zebrafish embryo. (A) Western blot analysis shows that PaTu-S but not PaTu-T cells express E-cadherin and both express α-, β- and γ-catenin. GAPDH expression is shown as a control. (B) Cellular localization of E-cadherin and β-catenin was analysed by immunofluoresscence. E-cadherin expression is shown for PaTu-S cells (a) and absence of E-cadherin expression for PaTu-T cells (b). Dapi staining was used to visualize cell nuclei in blue. β-catenin localization is shown for PaTu-S (c) and for PaTu-T (d). Co-localization of E-cadherin (green) and β-catenin (red) in PaTu-S cells is indicated by yellow staining of the plasmamembrane (e). (C) An in vitro migration assay (′scratch assay′) shows differences in migration of the two cell lines (PaTu-S: a-c and PaTu-T: d-f). Similar results were obtained in four independent experiments. Gap closure (gap width) over time is shown in Additional file 5. (D) Non-invasive PaTu-S cells (green) and invasive PaTu-T cells (red) were implanted consecutively in the same embryo (a and b) (Scales: 250 μm (a) and 300 μm (b)). (E) Homozygous cloche mutants [17] were injected with PaTu-T cells and followed over time. Shown is an example of a cloche^-/^- zebrafish at 3 dpi (scale bar: 300 μm). In contrast to control zebrafish none of the tested cloche^-/^-mutants showed any sign of metastatic behaviour (see Additional file 1 and Additional file 7). The cloche phenotype and its lack of a functional vasculature and circulation is observable by DIC microscopy (see Additional file 6).