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Protein Tyrosine Phosphatases pp 331–349Cite as

Functional Analysis of Dual-Specificity Protein Phosphatases in Angiogenesis

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1447))

Abstract

Therapeutic perspectives targeting angiogenesis in cancer stimulated an intense investigation of the mechanisms triggering and governing angiogenic processes. Several publications have highlighted the importance of typical dual-specificity phosphatases (DSPs) or MKPs in endothelial cells and their role in controlling different biological functions implicated in angiogenesis such as migration, proliferation, apoptosis, tubulogenesis, and cell adhesion. However, among atypical DSPs, the only one investigated in angiogenesis was DUSP3. We recently identified this DSP as a new key player in endothelial cells and angiogenesis. In this chapter we provide with detailed protocols and models used to investigate the role of DUSP3 in endothelial cells and angiogenesis. We start the chapter with an overview of the role of several DSPs in angiogenesis. We continue with providing a full description of a highly efficient transfection protocol to deplete DUSP3 using small interfering RNA (siRNA) in the primary human umbilical vein endothelial cells (HUVEC). We next describe the major assays used to investigate different processes involved in angiogenesis such as tube formation assay, proliferation assay and spheroids sprouting assay. We finish the chapter by validating our results in DUSP3-knockout mice using in vivo angiogenesis assays such as Matrigel plug and Lewis lung carcinoma cell subcutaneous xenograft model followed by anti-CD31 immunofluorescence and ex vivo aortic ring assay. All methods described can be adapted to other phosphatases and signaling molecules.

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Acknowledgement

This work was supported by grants from the Fonds de la Recherche Scientifique (FRS-FNRS) (Belgium), the Fonds Spéciaux pour la Recherche from the University of Liège (Belgium), from the Léon Fredericq Funds (FLF) and the “Centre Anti-cancéreux” (CAC). AM and EC are Télévie PhD fellows.

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Authors and Affiliations

  1. Immunology and Infectious Diseases Unit, GIGA-Signal Transduction, University of Liège, 1, Avenue de l’hôpital, B34., 4000, Liège, Belgium

    Mathieu Amand & Souad Rahmouni

  2. Laboratory of Tumor and Developmental Biology, GIGA-Cancer, University of Liège, Liège, Belgium

    Charlotte Erpicum, Christine Gilles & Agnès Noël

Authors
  1. Mathieu Amand
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  2. Charlotte Erpicum
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  3. Christine Gilles
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  4. Agnès Noël
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  5. Souad Rahmouni
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Corresponding author

Correspondence to Souad Rahmouni .

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Editors and Affiliations

  1. BioCruces Health Research Institute, Barakaldo, Bizkaia, Spain

    Rafael Pulido

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Amand, M., Erpicum, C., Gilles, C., Noël, A., Rahmouni, S. (2016). Functional Analysis of Dual-Specificity Protein Phosphatases in Angiogenesis. In: Pulido, R. (eds) Protein Tyrosine Phosphatases. Methods in Molecular Biology, vol 1447. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3746-2_18

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  • DOI: https://doi.org/10.1007/978-1-4939-3746-2_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3744-8

  • Online ISBN: 978-1-4939-3746-2

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