Activin-induced Growth Suppression http://www.selleckchem.com/products/Y-27632.html is Dependent on p21 Expression, and SMAD4/p21 Signaling can Counteract Activin-induced SMAD4-independent Migration Subsequently, we sought to determine the functional role of p21 in activin-induced growth suppression and cell viability. We found that loss of p21 via siRNA knockdown resulted in abolishment of activin-induced growth suppression in SMAD4-wild type cells, indicating that activin/SMAD4-induced growth suppression is p21-dependent (Figure 3A). Further, loss of p21 not only led to abrogation of activin-induced cell death, but also to an increase in cell numbers suggesting a survival benefit with loss of p21 (Figure 3B). These observations underscore the importance of the SMAD4/p21 axis in activin-mediated growth suppression and cell death.

Figure 3 p21 mediates activin-induced growth suppression and counteracts activin-induced SMAD4-independent migration in the presence of SMAD4. Accordingly, we tested the role of the p21 in activin-induced migration by assessing cellular mobility in the presence and absence of SMAD4. We found that activin enhanced migration in SMAD4 positive and SMAD4 negative cells (Figure 3C), which argues for a SMAD4 independent pathway regulating migration. This data is supported by similar findings in TGF�� signaling for which a strong pro-migratory and SMAD4-independent effect was shown [20], [22]. As activin treatment was associated with a decrease in p21 levels as well an increase in migration, we expected that loss of p21 by knockdown would enhance baseline migration as well as migration after activin treatment in SMAD4 intact cells, if the remaining p21 was at least partially involved in counteracting activin-induced migration.

Consistent with this hypothesis, we show an increased basal migration rate in SMAD4 expressing cells following p21 knockdown (Figure 3C) as well as overall more pronounced migration upon activin treatment (Figure 3D). We further found that basal cell migration was enhanced by activin treatment in the absence of either p21 or SMAD4 in SMAD4-positive FET cells, but that knockdown of p21 had no additional effect on migration when SMAD4 was absent, as in SW480 cells (Figure 3D). For TGF��, we found that cell migration was enhanced regardless of the presence of p21 (Figure 2B, Figure S1D).

This supports again that p21-mediated effects following activin treatment are dependent on SMAD4 and that p21 acts downstream of SMAD4 for its anti-proliferative and anti-migratory effects (Figure 4). Further, this suggests that, in the case of TGF��, some Dacomitinib promigratory signals can bypass SMAD4 as previously postulated [22] circumventing p21 and its inhibitory effects. In summary, we deduce that p21 may counteract migration downstream of SMAD4 and that downregulation of p21 may be responsible for some of the pro-migratory potential of activin signaling.