Figure 3.

Inhibitors of Mek-1 prevent and reverse EMT, and PI3K inhibitors abolish protection from TGFβ-induced apoptosis. (A) EpH4 cells (lanes marked E) or EpRas cells mock treated (lanes marked R) or treated with the inhibitors indicated (lanes marked iR) were lysed after 6 h of inhibitor treatment and analyzed for serine phosphorylation of Erk1/2 (MAPK) and PKB/Akt in Western blots (as described in Materials and methods). (B) EpRas cells seeded into collagen gels in the presence or absence (insets) of 5 ng/ml TGFβ. Both types of cultures were then left untreated (top, control) or treated with PD98059 (top right) and LY294002 (bottom) for another 5 d (as described in Materials and methods). Neither inhibitor was toxic to EpRas cells without TGFβ (insets; LY294002 slowed down structure growth). PD98059 (10 μM) completely reverted EMT (white arrows, top right), 5 μM LY294002 had no effect on the mesenchymal structures (black arrows, bottom left), whereas 30 μM caused cell death (bottom right, red circle). (C) Confocal analysis of in situ TUNEL staining (green) performed on collagen gel structures from EpRas cells treated for 4 d as indicated on the microphotographs (counterstaining for DNA in blue). (D) Quantitative analysis of cell death caused by inhibition of MEK-1 and PI3K in the absence or presence of TGFβ using trypan blue dye exclusion assay. The percentages of structures consisting of 25–60% (hatched bars, partially dead) or >60% trypan blue–positive cells (black bars, dead) are shown (as described in Materials and methods). Red asterisks indicate statistically significant induction of apoptosis by TGFβ. Bars: (B and C) 50 μm.