High-content imaging and analysis to quantify the nuclear to cytoplasmic ratio of TGFβ and hippo effectors in mammalian cells

Automated high-content immunofluorescence (IF) microscopy is used to monitor and quantify localization of the TGFβ/Smads and Taz/Yap Hippo effectors in mouse epithelial EpH4 cells transfected with Taz/Yap siRNAs. The nuclear-to-cytoplasmic protein ratios obtained by IF are converted into normalized...

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Autores principales: Labibi, Bita, Bashkurov, Mikhail, Christova, Tania, Attisano, Liliana
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2021
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8254078/
https://www.ncbi.nlm.nih.gov/pubmed/34258593
http://dx.doi.org/10.1016/j.xpro.2021.100632
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author Labibi, Bita
Bashkurov, Mikhail
Christova, Tania
Attisano, Liliana
author_facet Labibi, Bita
Bashkurov, Mikhail
Christova, Tania
Attisano, Liliana
author_sort Labibi, Bita
collection PubMed
description Automated high-content immunofluorescence (IF) microscopy is used to monitor and quantify localization of the TGFβ/Smads and Taz/Yap Hippo effectors in mouse epithelial EpH4 cells transfected with Taz/Yap siRNAs. The nuclear-to-cytoplasmic protein ratios obtained by IF are converted into normalized masses by estimating the ratio of the compartment volumes. This method has the advantage that endogenous rather than tagged proteins are tracked and that knockdown of Taz/Yap can be simultaneously monitored at the single-cell level. For complete details on the use and execution of this protocol, please refer to Labibi et al. (2020).
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spelling pubmed-82540782021-07-12 High-content imaging and analysis to quantify the nuclear to cytoplasmic ratio of TGFβ and hippo effectors in mammalian cells Labibi, Bita Bashkurov, Mikhail Christova, Tania Attisano, Liliana STAR Protoc Protocol Automated high-content immunofluorescence (IF) microscopy is used to monitor and quantify localization of the TGFβ/Smads and Taz/Yap Hippo effectors in mouse epithelial EpH4 cells transfected with Taz/Yap siRNAs. The nuclear-to-cytoplasmic protein ratios obtained by IF are converted into normalized masses by estimating the ratio of the compartment volumes. This method has the advantage that endogenous rather than tagged proteins are tracked and that knockdown of Taz/Yap can be simultaneously monitored at the single-cell level. For complete details on the use and execution of this protocol, please refer to Labibi et al. (2020). Elsevier 2021-06-28 /pmc/articles/PMC8254078/ /pubmed/34258593 http://dx.doi.org/10.1016/j.xpro.2021.100632 Text en © 2021 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Labibi, Bita
Bashkurov, Mikhail
Christova, Tania
Attisano, Liliana
High-content imaging and analysis to quantify the nuclear to cytoplasmic ratio of TGFβ and hippo effectors in mammalian cells
title High-content imaging and analysis to quantify the nuclear to cytoplasmic ratio of TGFβ and hippo effectors in mammalian cells
title_full High-content imaging and analysis to quantify the nuclear to cytoplasmic ratio of TGFβ and hippo effectors in mammalian cells
title_fullStr High-content imaging and analysis to quantify the nuclear to cytoplasmic ratio of TGFβ and hippo effectors in mammalian cells
title_full_unstemmed High-content imaging and analysis to quantify the nuclear to cytoplasmic ratio of TGFβ and hippo effectors in mammalian cells
title_short High-content imaging and analysis to quantify the nuclear to cytoplasmic ratio of TGFβ and hippo effectors in mammalian cells
title_sort high-content imaging and analysis to quantify the nuclear to cytoplasmic ratio of tgfβ and hippo effectors in mammalian cells
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8254078/
https://www.ncbi.nlm.nih.gov/pubmed/34258593
http://dx.doi.org/10.1016/j.xpro.2021.100632
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