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Open Access Highly Accessed Research article

Complete reversal of epithelial to mesenchymal transition requires inhibition of both ZEB expression and the Rho pathway

Shreyas Das1, Bryan N Becker3, F Michael Hoffmann12 and Janet E Mertz1*

Author Affiliations

1 McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, 1400 University Ave, Madison, Wisconsin 53706, USA

2 Laboratory of Genetics, University of Wisconsin School of Medicine and Public Health, 425-G Henry Mall, Madison, Wisconsin 53706, USA

3 Department of Medicine, University of Wisconsin School of Medicine and Public Health, 600 Highland Avenue, Madison, Wisconsin 53792, USA

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BMC Cell Biology 2009, 10:94  doi:10.1186/1471-2121-10-94

Published: 21 December 2009

Additional files

Additional File 1:

Higher dose of kinase inhibitors by themselves does not reverse stress fiber actin in mTEC-KO cells; rather, a combination of TβRI inhibitor and a ROCK inhibitor is required to reverse EMT. mTEC-KO cells were incubated with 100 pM TGF-β1 for 72 hours, kinase inhibitors were added, and incubation was continued for an additional 24 hours. F-actin was visualized by staining with Texas Red-phalloidin. Cells were viewed with an oil-objective lens at a 630× magnification. mTEC-KO cells were (A) untreated or treated with (B) 100 pM TGF-β1 for 72 hours followed by (C-E) single kinase inhibitor or (F-G) SB431542 plus a second kinase inhibitor. Single kinase inhibitors and concentrations were as follows: (C) 10 μM SB431542, (D) 10 μM SB203580, and (E) 10 μM Y27632. Combinations of kinase inhibitors were 10 μM SB431542 with (F) 10 μM SB203580 and (G) 10 μM Y27632. White arrows point to stress fibers.

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Additional File 2:

A combination of TβRI inhibitor and a ROCK inhibitor is required to reverse EMT in mTEC-WT cells. mTEC-WT cells were incubated with 100 pM TGF-β1 for 72 hours, kinase inhibitors were added, and incubation was continued for an additional 24 hours. F-actin was visualized by staining with Texas Red-phalloidin. mTEC-WT cells were (A) untreated or treated with (B) 100 pM TGF-β1 followed by (C) 10 μM SB431542 plus 10 μM Y27632. White arrows point to stress fibers.

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Additional File 3:

TGF-β1 induces ZEB1 and ZEB2 RNA accumulation in mTEC-KO cells. mTEC-KO cells were incubated for the times indicated with 100 pM TGF-β1. Cells were harvested and assayed by quantitative RT-PCR for ZEB1 and ZEB2 RNA. Data shown are means + S.E.M.s of two experiments performed in triplicate. Asterisk (*) indicates significant difference (P < 0.05, n = 6).

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