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Fig 1. Smad2 activation is stable under continuous stimulation. (A) Constructs used to generate the RFP-Smad2 cell line. Triangles denote the ePiggyBac terminal repeats, arrows denote promoters, and gray boxes denote antibiotic resistance genes. (B) Images of live untreated cells (Upper) or cells treated with 5 ng/mL TGF-β1 for 1 h (Lower) showing accumulation of RFP-Smad2 in the nucleus upon TGF-β1 treatment. (C) Quantification of average nuclear RFP-Smad2 from live cell imaging in cells exposed first to 5 ng/mL TGF-β1 and then exposed to 10 uM SB431542 1 h later, showing that pathway inhibition results in relocalization of RFP-Smad2 to the cytoplasm (D) Time courses of nuclear RFP-Smad2 show stable nuclear accumulation in cells exposed to either high (5 ng/mL) or low (0.1 ng/mL) doses of TGF-β1. Black lines represent averages of all cells in the field of view (>50 cells). Colored lines represent selected single cells. Cell nuclei were identified automatically and tracked through time using the GFP-NLS nuclear marker. (E) Immunofluorescent staining for Smad2/3 at the indicated times after continuous application of 1 ng/mL TGF-β1 showing stable enrichment of endogenous Smad2/3 in the nucleus. (F) Western blots of total cell lysates from cells continuously exposed to 5 ng/mL TGF-β1 for the indicated times show stably elevated pSmad2/3 levels Image published in: Warmflash A et al. (2012) Copyright © 2012. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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