FIGURE 4.

Stable expression of Smad3 restores the cell cycle arrest induced by TGF-β in EpRas cells. A, EpH4, EpRas, and EpRas S3 C1 cells were synchronized by contact inhibition and released by trypsinization into fresh medium in the presence or absence of TGF-β (2 ng/ml). Cells were collected at different time points and analyzed by FACS, to determine the number of cells in G₁, S, and G₂/M. Percentage of cells in each phase of the cell cycle are given. Cells were also collected for analysis by Western blotting (B). FLAG-Smad3 can be observed as a band running with a slightly lower mobility than endogenous Smad3 in extracts prepared from EpRas S3 C1 cells only. Grb2 serves as a loading control. C, cells were synchronized by contact inhibition then released by trypsinization into fresh medium with or without TGF-β in the presence of 10 mm BrdUrd. BrdUrd incorporation was measured by FACS analysis.