MTT-Growth Proliferation Assay Cellular metabolic activity, indicative of the growth status of cells following treatment with activin or TGF�� was assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (MP Biomedicals, Aurora, OH) as previously described [20]. Cell Death/Apoptosis TUNEL Assay/Cellular DNA Fragmentation ELISA Cells were http://www.selleckchem.com/products/BAY-73-4506.html seeded in 6 well plates at a density of 10,000 cells per well, serum starved for 24 hours and treated with ligand. 24 hours after treatment, cells were lysed with trypsin and counted using a hemacytometer as previously described [21]. Apoptosis was determined using TUNEL staining with APOPTAG In Situ Detection and DAPI counterstaining (Chemicon International/Millipore, Temecula, CA), according to the manufacturer��s instructions.

As an alternative assay of apoptosis, a photometric enzyme-linked immunoabsorbent assay for the detection of BrdU-labeled DNA fragments (Cellular DNA Fragmentation ELISA, Roche, Indianapolis, IN) was used. Both FET and SW480 cells were seeded in 6 well plates at a density of 10,000 cells per well. FET cells were seeded with or without SMAD4 or p21 siRNA. After 24 hours serum starvation, cells were treated with ligand for 24 hours. Apoptosis was determined via BrdU-labeling of intracellular DNA fragments and quantified via anti-DNA antibody detection ELISA according to the manufacturer��s guidelines. p21 Luciferase Assay The pWWP-luc plasmid (generous gift from B. Vogelstein, Johns Hopkins University, Baltimore, MD), containing the promoter of p21cip1/waf1, was cotransfected with the Renilla-expressing pRL-TK vector (Promega).

Luciferase activity was measured 24 hours after transfection by a dual luciferase reporter system (Promega). Relative luciferase activity was normalized to the Renilla luciferase activity as previously described [21]. Quantitative Expression of p21 mRNA RNA was extracted using the AllPrep DNA/RNA Mini Kit (Qiagen, Valencia, CA). RNA quality was assessed with the Agilent Bio-Chip (RIN >9.5). One microgram of RNA of each sample was reverse-transcribed using the Superscript III First-Strand Synthesis SuperMix and Oligo(dT)20 primers by Invitrogen according to the manufacturer��s instruction. Reverse transcription was followed by RNase H digest (New England Biolabs, Ipswich, MA). Quantitative PCR was carried out using specific primers for p21 (5��-GACTCTCAGGGTCGAAAACG-3��, 5��-GGATTAGGGCTTCCTCTTGG-3��).

Each experiment was performed as a standard curve experiment based on five serial dilutions (110), utilizing the Fast SYBR Green PCR MasterMix (Applied Biosystems, Foster City, CA). Each reaction was performed in triplicate using a total reaction volume of 20 ��l and a final primer concentration of 100 nM. The Brefeldin_A experiments were performed and analyzed on the 7900 HT Fast Real-Time PCR System (Applied Biosystems) following the standard protocol and conditions for the Fast SYBR Green Master Mix.