While TGFβ increases p21 expression in the presence of SMAD4, activin decreases nuclear and total p21 independent of SMAD4 status.

<p>A) <i>SMAD4</i>-wild type FET and <i>SMAD4</i>-null SW480 colon cancer cells were treated with vehicle (control), activin, or TGFβ for 24 hours. p21-specific transactivation was determined using a dual luciferase assay with pWWP-luc and pRL-TK (left panel) and mRNA expression levels of p21 were quantified by qPCR and normalized to L19 (right panel). While TGFβ markedly induced both p21-specific transactivation and transcription in the presence of SMAD4, no increase in p21 transactivation and only a modest increase in transcription following activin treatment in the presence of SMAD4 were found (*p<0.05). B) <i>SMAD4</i>-wild type FET and <i>SMAD4</i>-null SW480 cells were treated with control vehicle (C), activin (A), TGFβ(T), or a combination of both ligands (A+T) for 24 hours prior to lysis for total protein, nuclear, and cytoplasmic preparation. Histone H3, α-tubulin, and GAPDH were used as loading controls for the respective fractions. While TGFβ markedly increased p21 levels in all three fractions in the SMAD4 positive cell line only, activin induced a decrease in nuclear and total p21 protein in SMAD4-positive and -negative cells (left panel). Densitometric analysis of all blots revealed statistically significant changes in p21 levels (right panel) (ns = non-significant, *p<0.05, **p<0.01, ***p<0.001). C) Initial upregulation of p21 protein is followed by downregulation by 24 h after activin treatment. <i>SMAD4</i>-wild type FET cells were treated with activin or vehicle (control) and harvested at various time points for quantification of p21 protein expression. GAPDH was used as loading control and relative expression was calculated via densitometry. D) While TGFβ-induced upregulation of p21 was SMAD4 dependent, activin-induced downregulation of p21 was still observed in the absence of SMAD4. <i>SMAD4</i>-wild type FET cells were treated with vehicle (CNT), activin or TGFβ in the presence of either scramble siRNA (SC) or SMAD4 siRNA (KD) and total p21 levels were determined. GAPDH was used as loading and C32 cell lysate as p21 positive control.