Fig. 3

TGFβ but not activin stabilizes p21 via SMAD4 and MEK/ERK. a TGFβ-induced p21 upregulation is MEK/ERK dependent and not influenced by PI3K inhibition. In contrast, activin-induced p21 downregulation is dependent on PI3K signaling, but not dependent on SMAD4. ACVR2A/TGFBR2 wild type FET cells, FET with SMAD4 knockdown, and the SMAD4-null colon cancer cell line SW480 were treated with activin or TGFβ for 24 h 30 min after pharmacologic inhibition of PI3K (LY 290042) or MEK1/2 inhibition (U0126) and p21 levels determined by Western blot. GAPDH was used as a loading control. Three independent experiments entered the densitometric analysis shown below the representative blots (*p < 0.05, **p < 0.01, ***p < 0.001). For simplicity, we only show level of significance between control versus activin; control versus TGFβ; and activin versus LY and TGFβ versus U. b TGFβ treatment prominently increases the phosphorylation of ERK1/2 in colon cancer cells. Isoelectric point immunoassay was performed in FET cells treated with activin or TGFβ and pERK1/2 and ppERK1/2 compared to vehicle control. Five independent experiments were performed (*p < 0.05, **p < 0.01, ***p < 0.001). c TGFβ utilizes MEK/ERK to induce pSMAD2. ACVR2A/TGFBR2 wild type FET cells were treated with activin, TGFβ or vehicle control for 24 h following pretreatment with PI3K and MEK/ERK inhibition. pSMAD2 was determined by Western blot analysis and GAPDH was used as a loading control. Activin-induced pSMAD2 increase is independent from PI3K and MEK/ERK, but inhibition of MEK/ERK abolishes TGFβ-induced upregulation of pSMAD2 (A + LY Activin + LY; A + U Activin + U0126; T + LY TGFβ + LY; T + U TGFβ + U0126)