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.</p

Nuclear p21 expression in primary colon cancers correlates with ACVR2 and TGFBR2 receptor expression.

<p>A) ACVR2 expression correlates with loss of nuclear p21 in colorectal cancers:</p><p>χ²(1, N = 56) = 15.204, p = 0.0001.</p><p>B) Loss of TGFBR2 expression correlates with loss of nuclear p21 in colorectal cancers:</p><p>χ²(1, N = 56) = 11.755, p = 0.0006.</p

p21 mediates activin-induced growth suppression and counteracts activin-induced SMAD4-independent migration in the presence of SMAD4.

<p>A) FET cells were treated with either scramble (SC) or p21 specific siRNA (KD). Growth suppression was assessed by MTT-metabolic assay following activin treatment. Activin induced cell growth inhibition in the presence of p21, but the effect was reversed in the absence of p21 (*p<0.05). B) Total viability is decreased in SMAD4 wild type colon cancers following activin treatment in the presence of p21. FET cells were treated with either scramble or p21 specific siRNA. Cell viability was assessed by trypan blue staining following activin treatment. Trypan blue positive cells after activin treatment were decreased in presence of p21, but increased after p21 knockdown (***p<0.001). C) Activin (A) induces cell migration in SMAD4-positive and SMAD4-negative cell lines. Cellular migration is induced in <i>SMAD</i>4-wild type FET cells and <i>SMAD4</i>-null SW480 cells following activin treatment, but more pronounced induction of migration is seen in the absence of SMAD4. Loss of p21 leads to an increase in baseline migration in SMAD4 expressing cells (*p<0.05, **p<0.01, ***p<0.001). D) p21 knockdown increases the overall pro-migratory effect of activin in FET cells. Loss of p21 in the absence of SMAD4 does further increase migratory induction (*p<0.05, ** p<0.01).</p

Schematic of proposed differential regulation and effects of activin and TGFβ signaling on p21 in colon cancer cells. * is indicative of total (cytoplasmatic + nuclear) p21.

<p>Schematic of proposed differential regulation and effects of activin and TGFβ signaling on p21 in colon cancer cells. * is indicative of total (cytoplasmatic + nuclear) p21.</p

Expression of p21 is lost in a subset of primary colon cancers correlating with the ACVR2/TGFBR2 receptor status.

<p>Fifty-six colon cancers were stained for ACVR2, TGFBR2 and p21. Representative examples for p21 staining are shown: normal colon tissue with nuclear staining (left panel), colon cancer sample with maintained nuclear p21 staining (middle panel), and colon cancer sample with loss of nuclear p21 staining (right panel).</p

Activin-induced p21 downregulation is associated with ubiquitination and counteracted by proteasomal degradation.

<p>A) <i>ACVR2/TGFBR2/SMAD4</i>-wild type FET cells were were pretreated for 30 minutes with proteasomal inhibitor MG-132 and then treated with vehicle (control), activin, TGFβ for 24 hours and ubiquitination of total p21 was assessed via immunoprecipitation of p21 and blotting with a ubiquitin-specific antibody (upper panel) and reblotting of p21. Multiple bands indicative of polyubiquitination were seen only following activin treatment. B) Activin-induced p21 downregulation is dependent on the proteasome. <i>SMAD4</i>-wild type FET cells were pretreated for 30 minutes with proteasomal inhibitor MG-132 followed by treatment with vehicle (control) or activin for 24 hours and compared to cells treated accordingly without proteasomal inhibition. p21 expression was assessed and showed inhibition of p21 downregulation following activin treatment in conjunction with proteasomal inhibition.</p

Resveratrol Exerts Dosage and Duration Dependent Effect on Human Mesenchymal Stem Cell Development

<div><p>Studies in the past have illuminated the potential benefit of resveratrol as an anticancer (pro-apoptosis) and life-extending (pro-survival) compound. However, these two different effects were observed at different concentration ranges. Studies of resveratrol in a wide range of concentrations on the same cell type are lacking, which is necessary to comprehend its diverse and sometimes contradictory cellular effects. In this study, we examined the effects of resveratrol on cell self-renewal and differentiation of human mesenchymal stem cells (hMSCs), a type of adult stem cells that reside in a number of tissues, at concentrations ranging from 0.1 to 10 µM after both short- and long-term exposure. Our results reveal that at 0.1 µM, resveratrol promotes cell self-renewal by inhibiting cellular senescence, whereas at 5 µM or above, resveratrol inhibits cell self-renewal by increasing senescence rate, cell doubling time and S-phase cell cycle arrest. At 1 µM, its effect on cell self-renewal is minimal but after long-term exposure it exerts an inhibitory effect, accompanied with increased senescence rate. At all concentrations, resveratrol promotes osteogenic differentiation in a dosage dependent manner, which is offset by its inhibitory effect on cell self-renewal at high concentrations. On the contrary, resveratrol suppresses adipogenic differentiation during short-term exposure but promotes this process after long-term exposure. Our study implicates that resveratrol is the most beneficial to stem cell development at 0.1 µM and caution should be taken in applying resveratrol as an anticancer therapeutic agent or nutraceutical supplement due to its dosage dependent effect on hMSCs.</p> </div

Resveratrol exerts dosage dependent enhancing vs. inhibitory effect on the self-renewal rate of hMSCs.

<p><b>A</b>). Cells were plated at equal density and cultured in different concentrations of resveratrol continuously for 36 days during which cells were counted and split at equal ratio every 6 days. *: p<0.01. <b>B</b>). Cells pretreated with resveratrol or BM for 0 (0D-PT), 9 (9D-PT), 12 (12D-PT) or 30 (30D-PT) days were seeded at 8000 cells/well and continued to culture in corresponding media until resazurin assay. Error bars represent standard deviation (triplicates in each treatment condition). *: p<0.05 vs. BM.</p

Resveratrol regulates the expression of genes implicated in osteogenesis and adipogenesis.

<p><b>A</b>). Representative gel images of gene expression examined by semi-quantitative RT-PCR on cells subjected to concurrent treatment of BM/resveratrol with OIM for 3 days (OIM 3D-CT) or 7 days (OIM 7D-CT), or cells pretreated with BM/resveratrol for 12 days followed by 3 days of OIM (12D PT-3D OIM) or 7 days of AIM (12D PT–7D AIM) exposure. Expression of internal control gene <i>Hsp90</i> from the same batch of <i>cDNA</i> for each gene is shown in the bottom row. <b>B</b>). Expression of each gene in resveratrol treated cells was quantified relative to that in BM treated cells and normalized by the expression of housekeeping gene <i>Hsp90</i>. Data shown are the mean values of three repeats. Error bars represent standard deviation. Concentration unit: µM. *: p<0.05 vs. BM. **: p = 0.055 vs. BM.</p

Resveratrol exerts dosage dependent anti- vs. pro-senescence effect on hMSCs.

<p><b>A</b>). Cells were stained in X-gal (blue color) and neutral red solution (red color). Images were taken at 200× magnification. <b>B</b>). Percentages of senescent cells vs. total cells were determined based on images taken. At least 184 total cells from each treatment condition were counted. Column represents the relative fold changes of percentage of cells undergoing senescence in each treatment group normalized to the value obtained in the BM treated cells. Data was obtained from three independent experiments. Error bars represent standard deviation. *: p≤0.01 vs. BM.</p