Fig. 8. Activation of the JNK pathway by WNT3a is not obligate for formation of primitive endoderm. F9 cells stably expressing rFZ1 were treated with a JNK inhibitor (0.4 µM SP600125) or transiently transfected with DN-RhoA or DN-MEKK 1 before stimulation with WNT3a for 4 days. Subsequently, the cells were prepared for immunocytochemistry and stained with a monoclonal antibody against the cytokeratin endo A (TROMA-1), a marker protein for primitive endoderm. Alexa-Fluor-488-conjugated secondary antibodies were employed together with indirect epifluorescence to detect the immune complexes. Typical phase-contrast images (PC) and the indirect immunofluorescence images (IIF) are shown from a single experiment, representative of three independent experiments.