title: Functional Analysis of Dual-Specificity Protein Phosphatases in Angiogenesis.
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.
authors: Immunology and Infectious Diseases Unit, GIGA-Signal Transduction, University of Liège, 1, Avenue de l'hôpital, B34., 4000, Liège, Belgium.
authors: Mathieu
authors: M
authors: Amand
authors: Laboratory of Tumor and Developmental Biology, GIGA-Cancer, University of Liège, Liège, Belgium.
authors: Charlotte
authors: C
authors: Erpicum
authors: Laboratory of Tumor and Developmental Biology, GIGA-Cancer, University of Liège, Liège, Belgium.
authors: Christine
authors: C
authors: Gilles
authors: Laboratory of Tumor and Developmental Biology, GIGA-Cancer, University of Liège, Liège, Belgium.
authors: Agnès
authors: A
authors: Noël
authors: Immunology and Infectious Diseases Unit, GIGA-Signal Transduction, University of Liège, 1, Avenue de l'hôpital, B34., 4000, Liège, Belgium. srahmouni@ulg.ac.be.
authors: Souad
authors: S
authors: Rahmouni
mesh_terms: D000818:Animals; D018827:Carcinoma, Lewis Lung / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D054640:Dual Specificity Phosphatase 3 / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009389:Neovascularization, Pathologic / Q000378:metabolism; D018919:Neovascularization, Physiologic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D018874:Spheroids, Cellular / Q000166:cytology / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D034741:RNA, Small Interfering; C517025:DUSP3 protein, human; D054640:Dual Specificity Phosphatase 3; C517026:Dusp3 protein, mouse
keywords: Angiogenesis; Aortic ring assay; DSPs; DUSP3; Endothelial cells; HUVEC; Immunofluorescence; LLC; Matrigel plug assay; Proliferation; Spheroid assay; Sprouting