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

Maier J, Traenkle B, Rothbauer U - Sci Rep (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Dose-dependent reduction of vimentin upon Withaferin A treatment.A549_VB6-CB cells were treated with increasing concentrations of Withaferin A (WFA) in absence (−TGF) or presence of TGF-β (+TGF) and subjected to time-lapse microscopy for 24 h. The effect of WFA-treatment on vimentin fiber segments was quantified in >100 cells after 0 h, 3 h, 6 h, 12 h and 24 h. Shown are relative numbers of segments per cell representing the means ± s.e.m. of three independent experiments. Statistical analysis of the dose-dependency was performed using student’s t-test with reference to the untreated control (0 nM WFA), *P < 0.05, **P < 0.01, ***P < 0.001.
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f6: Dose-dependent reduction of vimentin upon Withaferin A treatment.A549_VB6-CB cells were treated with increasing concentrations of Withaferin A (WFA) in absence (−TGF) or presence of TGF-β (+TGF) and subjected to time-lapse microscopy for 24 h. The effect of WFA-treatment on vimentin fiber segments was quantified in >100 cells after 0 h, 3 h, 6 h, 12 h and 24 h. Shown are relative numbers of segments per cell representing the means ± s.e.m. of three independent experiments. Statistical analysis of the dose-dependency was performed using student’s t-test with reference to the untreated control (0 nM WFA), *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: To trace the effect of WFA on cells comprising different levels of vimentin, A549_VB6-CB cells were either left untreated or stimulated with TGF-β for 16 h prior to incubation with increasing concentrations of WFA. Subsequently, we monitored the chromobody signal continuously for 24 h. High-content imaging revealed a time and dose-dependent reduction of vimentin fiber segments upon incubation with WFA under both conditions (Fig. 6, Supplementary Fig. 8). Compared to the untreated control (0 nM), incubation with 250 nM and 500 nM WFA led to a significant reduction of vimentin fiber segments after 3 h, 6 h and 12 h (Fig. 6, Supplementary Fig. 8). Moreover, after 6 h and 12 h of WFA treatment, vimentin fiber segments were maximally reduced, whereas after 24 h the number of vimentin fiber segments almost completely recovered to initial levels independently of the WFA concentration (Supplementary Fig. 8). In the presence of TGF-β, treatment with WFA resulted in a two-fold reduction of vimentin within 12 h, while without TGF-β, we observed a ten-fold reduction within the same time period (Fig. 6). Taken together, these data demonstrate that high-content imaging of our vimentin chromobody cell model allows a precise quantification of dose and time-dependent compound effects independently of the initial level of vimentin expression.


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

Maier J, Traenkle B, Rothbauer U - Sci Rep (2015)

Dose-dependent reduction of vimentin upon Withaferin A treatment.A549_VB6-CB cells were treated with increasing concentrations of Withaferin A (WFA) in absence (−TGF) or presence of TGF-β (+TGF) and subjected to time-lapse microscopy for 24 h. The effect of WFA-treatment on vimentin fiber segments was quantified in >100 cells after 0 h, 3 h, 6 h, 12 h and 24 h. Shown are relative numbers of segments per cell representing the means ± s.e.m. of three independent experiments. Statistical analysis of the dose-dependency was performed using student’s t-test with reference to the untreated control (0 nM WFA), *P < 0.05, **P < 0.01, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4544033&req=5

f6: Dose-dependent reduction of vimentin upon Withaferin A treatment.A549_VB6-CB cells were treated with increasing concentrations of Withaferin A (WFA) in absence (−TGF) or presence of TGF-β (+TGF) and subjected to time-lapse microscopy for 24 h. The effect of WFA-treatment on vimentin fiber segments was quantified in >100 cells after 0 h, 3 h, 6 h, 12 h and 24 h. Shown are relative numbers of segments per cell representing the means ± s.e.m. of three independent experiments. Statistical analysis of the dose-dependency was performed using student’s t-test with reference to the untreated control (0 nM WFA), *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: To trace the effect of WFA on cells comprising different levels of vimentin, A549_VB6-CB cells were either left untreated or stimulated with TGF-β for 16 h prior to incubation with increasing concentrations of WFA. Subsequently, we monitored the chromobody signal continuously for 24 h. High-content imaging revealed a time and dose-dependent reduction of vimentin fiber segments upon incubation with WFA under both conditions (Fig. 6, Supplementary Fig. 8). Compared to the untreated control (0 nM), incubation with 250 nM and 500 nM WFA led to a significant reduction of vimentin fiber segments after 3 h, 6 h and 12 h (Fig. 6, Supplementary Fig. 8). Moreover, after 6 h and 12 h of WFA treatment, vimentin fiber segments were maximally reduced, whereas after 24 h the number of vimentin fiber segments almost completely recovered to initial levels independently of the WFA concentration (Supplementary Fig. 8). In the presence of TGF-β, treatment with WFA resulted in a two-fold reduction of vimentin within 12 h, while without TGF-β, we observed a ten-fold reduction within the same time period (Fig. 6). Taken together, these data demonstrate that high-content imaging of our vimentin chromobody cell model allows a precise quantification of dose and time-dependent compound effects independently of the initial level of vimentin expression.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Pharmaceutical Biotechnology, Eberhard Karls University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus