Figure 2.

Ras activity is required for induction and maintenance of EMT. (A–D) EpXT cells were seeded into collagen gels in the absence (A) or presence (B) of 10 μM Ras farnesylation inhibitor (L739749), and resulting structures were photographed after 6 d (as described in Materials and methods). Note the complete reversal of EpXT cells to tubular structures with lumina (white arrows), which persisted after removal of the inhibitor for 4 d (C). The inhibitor alone did not affect tubular structures formed by EpRas cells (B, inset) or after reversal (D, inset, control), whereas treatment of the reverted structures with TGFβ plus L739749 caused cell disintegration. (E and F) Immunostaining of frozen ultrathin sections of EpXT collagen gel structures before (E) or after treatment (F) with L739749 for 5 d. Sections were stained with antibodies to TGFβ (red) (Oft et al., 1996) and vimentin (green) plus DAPI (top, DNA). Parallel sections (bottom) were stained with antibodies to ZO-1 (red) and fibronectin (green). L739749 causes loss of intracellular TGFβ and vimentin (top) and extracellular fibronectin (bottom) and reexpression of ZO-1 at apicolateral sites of tight junctions (blue arrows, bottom left). Bars: (A–D) 50 μm; (E and F) 20 μm.