For normalization purposes expression levels of L19 were determined accordingly in the same run to exclude effects of inter-run variability (5��-ACCCCAATGAGACCAATGAAAT-3��, selleck chemicals llc 5��-CAGCCCATCTTTGATGAGCTT-3��). The relative expression of p21 normalized to L19 levels was calculated for each sample and plotted on a graph. Total Lysis, Nuclear/Cytosolic Separation, and Western Blotting Cells were lysed using total lysis buffer RIPA (1% NP40, 0.1% SDS, 1% DCA, 50 mM Tris HCl pH 7.2) with added protease and phosphatase inhibitors as previously described [20], [21]. Cytoplasmic and nuclear fractions were extracted with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer��s instruction.

Western blotting was performed using standard protocols with 4�C20% polyacrylamide gels, nitrocellulose membrane transfers, overnight incubation with primary antibody at 4��C followed by horseradish peroxidase-linked secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and detection by ECL (Amersham, Little Chalfont, UK) [20], [21], visualization, and quantification of chemiluminescence with the LAS-3000 (FujifilmUSA, Valhalla, NY). siRNA and Transfection Two specific siRNAs for each p21 and SMAD4 (Ambion, Austin, TX and Santa Cruz Biotechnology) were transiently delivered at a final concentration of 10 nM via electroporation using the AMAXA Nucleofector (Lonza, Basel, Switzerland) in 12-well plates at a density of 2��106 according to the manufacturer��s instructions. Transfection efficiency was confirmed using the pmaxGFP? Control Vector (Lonza).

Forty-eight hours post transfection, colon cancer cells were lysed for subsequent RNA and protein extraction. Migration/Invasion Assay Migration assays were performed as previously described [20]. Briefly, Corning Costar Transwell 12 well plates (8 ��m pores, Corning, NY) with fibronectin or matrigel (Sigma, St. Louis, MO) were seeded with colon cancer cells with or without ligand in the presence or absence of siRNA. Cells were then allowed to migrate for 4 hours, stained, and images were captured using an Axiovert 2000 microscope with an AxioCAM HRC Camera (both Zeiss Microimaging, Thornwood, NY). Images from 5 microscopic fields at the center of each well were counted.

Immunohistochemistry for ACVR2, TGFBR2, p21 Expression and Localization Slides containing primary colon cancer tissues were processed as previously described [6] and stained for ACVR2, TGFBR2, and p21 using the Catalyzed Signal Amplification Brefeldin_A System (CSA) by DAKO (Carpinteria, CA). ACVR2 and TGFBR2 staining was grouped into negative (no or weak signal) and positive (moderate or strong signal) receptor status. The percentage of p21 positive nuclei in each cancer samples was assessed. Tumors with more than 50% of p21 positive nuclei were scored as nuclear positive cancers.