LIF withdrawal induces a morphological change in ES-D3 cells from multilayered clusters to epithelial monolayers and impairs ES-D3 adhesion to collagen. A) ES-D3 cells were cultured for 5 days on tissue-culture dishes coated with LN-511, LN-111, Col-I, Col-IV or FN in the presence or absence of 10 ng/ml of LIF and imaged using a phase contrast microscope equipped with a CCD-camera. Scale bar is 100 μm. B) ES-D3 cells were cultured on LN-511 in the presence or absence of LIF or on LN-111, Col-I, Col-IV or FN in the absence of LIF, fixed with 4% PFA and stained for an epithelial AJ marker E-Cad (red) and a TJ marker ZO-1 (green). Cells were imaged using confocal microscopy and a single optical slice at the level of TJs (as determined by chicken-wire-like ZO-1 staining) is shown. Scale bar is 50 μm. C) ES-D3 cells were counted and seeded (1000 cells/mm2) onto tissue culture dishes (3.5 cm Ø) coated with the indicated ECM substrates and individual cells were tracked at 2 minutes intervals upon their contact with the ECM. The amount of adherent (immobile) cells was determined as described in materials and methods. The data shown comes from 2 independent experiments in which at least 50 individual cells were tracked. The relative amount of immobilized cells is depicted with differentially sized red circles the center of the graphs. D) A graph showing the percentage of ES-D3 cells immobilized on the indicated substrates within the first 30 minutes upon seeding.