Changes of migratory and invasive properties of Py2T cells before, during and after TGFβ-induced EMT.

<p>(<b>A</b>) Boyden chamber migration and invasion assay. Cells were treated with TGFβ for the indicated times (LT = long term treatment, as described in Fig. 1F). 25'000 cells were seeded into migration or invasion chambers in duplicate in the absence or presence of TGFβ and allowed to pass through the membrane pores for 24 hours along an FBS gradient. Invasion chambers were pre-coated with growth-factor reduced Matrigel (BD BioCoat chambers). Cells that passed through the membrane pores were stained with crystal violet and photographed (<i>bottom panels</i>) and then counted (<i>top graphs</i>). Results are expressed as mean ± S.E.M of three independent experiments. (<b>B</b>) Scratch wound healing assay. Cells pre-treated with TGFβ or not as indicated were starved over night and scratch wounds were introduced into confluent monolayers. Scratch wound closure was monitored by an IncuCyte™ live cell imaging system. Black masking represents initial gap width at 0 hours. Note the collective, sheet-like wound closure by untreated Py2T cells in contrast to single cell wound infiltration of TGFβ-treated cells (also see Movies S1 and S2 for live imaging data of this experiment). (<b>C</b>) Morphology of epithelial Py2T cells and mesenchymal Py2T LT cells grown on plastic tissue culture dishes (2D) and in Matrigel (4 mg/ml; 3D). Structures were grown for 6 days, and stained directly in Matrigel with antibodies against epithelial E-cadherin and ZO-1 or against mesenchymal vimentin and fibronectin. Immunofluorescence images were acquired by confocal microscopy. Scale bars, 25 µm. (<b>D</b>) Three-dimensional reconstruction of confocal imaging stacks from cells grown in Matrigel as described in (A) (See also Movies S5 and S6 for rotating 3D models). Scale bars, 25 µm.