Supporting Materials and Methods
Cell Culture, RNA Extraction, and Immunoblotting.
Four fibroblast lines were derived from Smad2-knockout (S2KO) and WT littermate (S2WT) mouse embryos (day 10.5) and Smad3-knockout (S3KO) and WT littermate (S3WT) mouse embryos (day 17) as described in detail (1). Experiments were performed between passages 15 and 22. Cells were grown to confluence in 10% FBS-containing medium, serum-starved (FBS 0.2%) for 24 hr before stimulation with 8 ng/ml transforming growth factor b (TGF-b ) (R & D Systems). In S2WT fibroblasts, TGF-b was used in the absence or presence of 10 µM mitogen-activated protein kinase kinase/extracellular signal-regulated kinase inhibitor U0126 (Promega), which was added 20 min before TGF-b . Cells were lysed in Trizol reagent (Invitrogen), and total RNA was extracted, purified, and quality-controlled as described (2). For immunoblotting, cells were prepared and treated exactly as described above before lysis in radioimmunoprecipitation assay buffer containing phosphatase inhibitors as described (2). Primary antibodies included anti-Smad2 (Zymed), anti-Smad3 (Zymed), anti-Smad4 B8 (Santa Cruz Biotechnology), anti-Smad7 (gift of Peter ten Dijke, Netherlands Cancer Institute, Amsterdam), anti-phospho Smad2 (Upstate Biotechnology, Lake Placid, NY), anti-phospho Smad3 (kind gift of Michael Reiss, Robert Wood Johnson Medical School/University of Medicine and Dentistry of New Jersey, New Brunswick, NJ), and anti-p42/44 and anti-phospo p42/p44 (Cell Signaling Technologies, Palo Alto, CA).Clustering and Principal Component Analysis.
Hierarchical cluster dendrograms were generated with TIGR MULTIEXPERIMENT VIEWER software (The Institute for Genomics Research, Rockville, MD) by using Euclidean distance metrics and bootstrapping protocols for resampling. Self-organizing maps were computed with Pearson correlations as a dissimilarity measure. Principal component analyses were performed by using Euclidean distance or Covariance metric as implemented in TIGR MULTIEXPERIMENT VIEWER or J-EXPRESS software (MolMine, Bergen, Norway), respectively.1. Piek, E., Ju, W. J., Heyer, J., Escalante-Alcalde, D., Stewart, C. L., Weinstein, M., Deng, C., Kucherlapati, R., Böttinger, E. P. & Roberts, A. B. (2001) J. Biol. Chem. 276, 19945–19953
2. Zavadil, J., Bitzer, M., Liang, D., Yang, Y. C., Massimi, A., Kneitz, S., Piek, E. & Böttinger, E. P. (2001) Proc. Natl. Acad. Sci. USA 98, 6686–6691.