The Lectin-Like Domain of Thrombomodulin Inhibits β1 Integrin-Dependent Binding of Human Breast Cancer-Derived Cell Lines to Fibronectin
Abstract
:1. Introduction
2. Materials and Methods
2.1. Construction of a Recombinant Human TM (Domains 1, 2, and 3)-Immunoglobulin Fc Fusion Protein (TMD123-Fc) Expression Vector
2.2. Expression and Purification of TM Domain-Fc Fusion Proteins
2.3. V-Well Cell Adhesion Assay
2.4. Cell Culture
2.5. β1. Integrin KO Cell with CRISPR/CAS9 System
2.6. Enzyme-Linked Immunosorbent Assay (ELISA)
2.7. Flow Cytometry Analysis
2.8. Antibodies Targeting Integrins
2.9. Cell Adhesion Experiments Using Fibronectin-Coated Fluorescent Beads
2.10. Statistical Analysis
3. Result
3.1. The Lectin-Like Extracellular Domain of TM Binds Fibronectin
3.2. Binding of Human Breast Cancer-Derived Cell Lines to Fibronectin Is Inhibited by TMD123-Fc in a Concentration-Dependent Manner
3.3. The Lectin-Like Domain of TM Inhibits the Binding of β1 Integrin from Human Breast Cancer-Derived Cell Lines to Fibronectin
3.4. TM Inhibits the Integrin-Dependent Binding of MDA-MB-231 to Fibronectin in the Presence of Ca2+
3.5. Binding of MDA-231 Cells to Fibronectin Is Inhibited by TMD123-Fc
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Kawamoto, E.; Nago, N.; Okamoto, T.; Gaowa, A.; Masui-Ito, A.; Akama, Y.; Darkwah, S.; Appiah, M.G.; Myint, P.K.; Obeng, G.; Ito, A.; Caidengbate, S.; Esumi, R.; Yamaguchi, T.; Park, E.J.; Imai, H.; Shimaoka, M. The Lectin-Like Domain of Thrombomodulin Inhibits β1 Integrin-Dependent Binding of Human Breast Cancer-Derived Cell Lines to Fibronectin. Biomedicines 2021, 9, 162. https://doi.org/10.3390/biomedicines9020162
Kawamoto E, Nago N, Okamoto T, Gaowa A, Masui-Ito A, Akama Y, Darkwah S, Appiah MG, Myint PK, Obeng G, Ito A, Caidengbate S, Esumi R, Yamaguchi T, Park EJ, Imai H, Shimaoka M. The Lectin-Like Domain of Thrombomodulin Inhibits β1 Integrin-Dependent Binding of Human Breast Cancer-Derived Cell Lines to Fibronectin. Biomedicines. 2021; 9(2):162. https://doi.org/10.3390/biomedicines9020162
Chicago/Turabian StyleKawamoto, Eiji, Nodoka Nago, Takayuki Okamoto, Arong Gaowa, Asami Masui-Ito, Yuichi Akama, Samuel Darkwah, Michael Gyasi Appiah, Phyoe Kyawe Myint, Gideon Obeng, Atsushi Ito, Siqingaowa Caidengbate, Ryo Esumi, Takanori Yamaguchi, Eun Jeong Park, Hiroshi Imai, and Motomu Shimaoka. 2021. "The Lectin-Like Domain of Thrombomodulin Inhibits β1 Integrin-Dependent Binding of Human Breast Cancer-Derived Cell Lines to Fibronectin" Biomedicines 9, no. 2: 162. https://doi.org/10.3390/biomedicines9020162