Phosphoproteomic Analysis and Organotypic Cultures for the Study of Signaling Pathways

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Signaling pathways are involved in key cellular functions from embryonic development to pathological conditions, with a pivotal role in tissue homeostasis and transformation. Although most signaling pathways have been intensively examined, most studies have been carried out in murine models or simple cell culture. We describe the dissection of the TGF-β signaling pathway in human tissue using CRISPR-Cas9 genetically engineered human keratinocytes (N/TERT-1) in a 3D organotypic skin model combined with quantitative proteomics and phosphoproteomics mass spectrometry. The use of human 3D organotypic cultures and genetic engineering combined with quantitative proteomics and phosphoproteomics is a powerful tool providing insight into signaling pathways in a human setting. The methods are applicable to other gene targets and 3D cell and tissue models.

OriginalsprogEngelsk
Artikelnummere4941
TidsskriftBio-protocol
Vol/bind14
Udgave nummer4
ISSN2331-8325
DOI
StatusUdgivet - 2024

Bibliografisk note

Funding Information:
The work was supported by the European Commission (GlycoSkin H2020-ERC), European Commission (Imgene H2O20), Lundbeck Foundation, The Danish Research Councils (Sapere Aude Research Leader grant to H.H.W.), Danish National Research Foundation (DNRF107), the Friis Foundation (H.H.W AND S.D), Danish Strategic Research Council, and the program of excellence from the University of Copenhagen (CDO2016). We thank Karin Uch Hansen, Birgit Poulsen, Karen Biré, and Louise Rosgaard Duus for the excellent technical assistance. We acknowledge the Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen. We would like to thank James G. Rheinwald, Harvard Institute of Medicine, for providing the N/TERT-1 cells. This work was described in Ye et al. [11] and Dabelsteen et al. [2].

Publisher Copyright:
© 2024 Bio-protocol LLC. All rights reserved.

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