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

Bita Labibi; Mikhail Bashkurov; Tania Christova; Liliana Attisano

STAR Protocols (Sep 2021)

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

Bita Labibi,
Mikhail Bashkurov,
Tania Christova,
Liliana Attisano

Affiliations

Bita Labibi: Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada; Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada
Mikhail Bashkurov: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada
Tania Christova: Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada; Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada
Liliana Attisano: Department of Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada; Donnelly Centre, University of Toronto, Toronto, ON M5S 3E1, Canada; Corresponding author

Journal volume & issue: Vol. 2, no. 3
p. 100632

Abstract

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Summary: 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).

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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