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. 2011 May 15;22(10):1686–1698. doi: 10.1091/mbc.E11-02-0103

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© 2011 Gregory et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — Autocrine TGF-β signaling is required for the maintenance of a mesenchymal state. (A) Real-time PCR measurements of TGF-β family members after exogenous TGF-β1 treatment of MDCK cells. TGF-β3 levels were not detected (N.D.) in untreated MDCK cells. Data are normalized to untreated levels for TGF-β1 and TGF-β2 or Day 2 levels for TGF-β3. (B) TGF-β1 and TGF-β2 secreted by MDCK and MDCK-TGF cells into the culture medium was measured by ELISA. A TGF-β3 ELISA was not available for measurement of secreted TGF-β3. (C) Real-time PCR measurement of ZEBs in MDCK-TGF cells after inhibition of TGF-β signaling using the TGF-βRI inhibitor (1 μM SB-505124; 13 d) (D) Immunofluorescence staining of E-cadherin (red) and ZO-1 (green), and rhodamine phalloidin staining of F-actin in MDCK-TGF cells either untreated or treated with a TGF-βRI inhibitor [as in (C)] or a TGF-β1/2/3 neutralizing antibody (100 μg/ml; 15 d). Cells are counterstained with DAPI to visualize nuclei. Scale bars = 50 μm. (E) Quantitation of miR-200 levels in MDCK-TGF cells treated with individual TGF-β1, -β2, -β3 and -β1/2/3 neutralizing antibodies (100 μg/ml) after 7 or 15 d of treatment. Values are the mean ± SD of three replicate measurements.