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Sci Rep. 2015 Aug 21;5:13402. doi: 10.1038/srep13402.

Real-time analysis of epithelial-mesenchymal transition using fluorescent single-domain antibodies.

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Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.
Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstr. 55, 72770 Reutlingen, Germany.


Vimentin has become an important biomarker for epithelial-mesenchymal transition (EMT), a highly dynamic cellular process involved in the initiation of metastasis and cancer progression. To date there is no approach available to study endogenous vimentin in a physiological context. Here, we describe the selection and targeted modification of novel single-domain antibodies, so-called nanobodies, to trace vimentin in various cellular assays. Most importantly, we generated vimentin chromobodies by combining the binding moieties of the nanobodies with fluorescent proteins. Following chromobody fluorescence in a cancer-relevant cellular model, we were able for the first time to monitor and quantify dynamic changes of endogenous vimentin upon siRNA-mediated knockdown, induction with TGF-β and modification with Withaferin A by high-content imaging. This versatile approach allows detailed studies of the spatiotemporal organization of vimentin in living cells. It enables the identification of vimentin-modulating compounds, thereby providing the basis to screen for novel therapeutics affecting EMT.

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