Both investigators had to be in agreement for a tumor to be called negative. Immunoprecipitation, Ubiquitination Assay, and Proteasomal Inhibition Proteasomal inhibition was achieved by pre-treatment of cells with 10 ��M of MG-132 (Calchemie) for 30 minutes. 80 ��g of total protein lysate from various treatments were incubated with 1 mg of p21 antibody Palbociclib purchase (# sc-469, Santa Cruz Biotechnology) overnight at 4��C. Protein G beads (Invitrogen, Carlsbad, CA) were added for 4 hours. After denaturation with sample buffer, Western blotting was performed on 4�C20% gradient gels (Biorad, Hercules, CA) and nitrocellulose membranes after boiling for 15 minutes incubated with antibodies against ubiquitin (11000) (# sc-8017) antibody (Santa Cruz Biotechnology), p21 and IgG (# 111-035-008; Jackson ImmunoResearch, West Grove, PA) or GAPDH (# sc-47724; Santa Cruz Biotechnology) as respective controls.
Statistical Analysis Differences between two respective groups were determined using Student��s t-test. Probability values less than 0.05 were considered to be significant. Data shown represents repeated experiments on multiple biological replicates. For association of p21 localization with receptor status, we performed Chi-Square test calculations using the MedCalc? software (Version 11.6.1). Supporting Information Figure S1 p21 mediates TGF��-induced growth suppresion and counteracts TGF��-induced SMAD4-independent migration in the presence of SMAD4. A) FET cells were treated with either scramble (SC) or p21 specific siRNA. Growth suppresion was assessed by MTT-metabolic assay following TGF�� treatment.
TGF�� induced cell grwoth inhibition in the presence of p21, but the effect was reversed in the absence of p21. B) Total viability is decreased in SMAD4-wild type colon cancer cells following TGF�� treatment in the presence of p21. FET cells were treated with either scramble (SC) or p21 specific siRNA. Cell viability was assessed by trypan blue staining following TGF�� treatment. Trypan blue positiv cells after TGF�� treatment were decreased in presence of p21, but increased after p21 knockdown. C) TGF�� induced cell migration in SMAD4-positiv and SMAD4-negativ cell lines. Cellular migration is induced in SMAD4-wild type FET cells and SMAD4-null SW480 cells following TGF�� treatment, but more pronounced induction of migration is seen in the absence of SMAD4.
D) p21 knockdown increased TGF��-induced migration in FET cells. Loss of in the absence of SMAD4 does not further increase migratory induction (*p<0.05, **p<0.01, ***p<0.001). (TIF) Click here for additional data file.(364K, tif) Table S1 Characteristics of colon Drug_discovery cancer patient cohort randomly selected from North Carolina Colorectal Cancer Study (NCCCS) (19, 20) (patients 1�C15) and NW cohort (patients 16�C56) for p21 staining. Four patients from the NW cohort did not have a stage information available (X). (DOC) Click here for additional data file.(92K, doc) Acknowledgments We thank Dr. John M.