Abstract
Abstract Aim NIH3T3 cell line with expression of human receptor for advanced glycation end-products (hRAGE) transduced with lentivirus vectors was used to analyze affinity, biological activity, and/or molecular mechanisms of molecules targeting the hRAGE pathway. Method The DNA fragment coding for hRAGE gene was integrated into the genome of NIH3T3 cells using lentivirus transduction. Cells expressing hRAGE were selected with puromycin, and the level of hRAGE expression was analyzed by Western blot. To establish a stable cell line, colonies of hRAGE-expressing cells were generated, and the level of RAGE expression in each engineered cell line was analyzed within 20 generations. Flow cytometry assay was used to verify affinity of anti-hRAGE antibody binding to hRAGE on the surface of engineered cells. The engineered NIH3T3 cell line was applied to assess effects of anti-hRAGE blocking antibody on amyloid β-induced cells apoptosis by CCK-8 assay. Results The engineered NIH3T3 cell line (hRAGE-NIH3T3) could stably express human RAGE. Commercial anti-RAGE polyclonal antibody could recognize and bind to human RAGE on the surface of hRAGE-NIH3T3 but not original NIH3T3 cells. In addition, hRAGE-NIH3T3 was more sensitive to RAGE pathway-dependent stimulation. Our data show that the hRAGE-NIH3T3 cell line established is an excellent tool in the study of RAGE-targeting molecules based on the cellular level, biological function, and RAGE-mediated molecular mechanisms.
Highlights
Receptor for advanced glycation end-products (RAGE) is one of the members of the immunoglobulin superfamily and a multiligand cell surface receptor with structural homology to other immunoglobulin-like receptors.[1]
Materials NIH3T3 and HEK293T were purchased from Chinese Academy of Sciences Cell Bank (ACSCB), anti-human receptor for advanced glycation endproducts (hRAGE) rabbit polyclonal antibody and rabbit immunoglobulin G (IgG) from Shanghai Kaijing Biotechnology Company, China, pLVX-puro-humanRAGE-HA from Universal Biological System Co., Ltd., China, Aβ1–42 from Chutai Biotechnology Company, China, mouse anti-HA tag antibody from Proteintech, United States, mouse anti-GAPDH antibody from Proteintech, United States, fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (HþL) and HRP-conjugated rabbit anti-mouse IgG (HþL) from Jackson, United States; CCK8 Kit from Dojindo, Japan, and substrate of enhanced chemiluminescence (ECL) from Merck Millipore, United States
Selection and Identification of hRAGE-NIH3T3 Cell Colonies Lentivirus is a useful tool in gene delivery and has high efficiency to integrate exogenous gene in the genome of host cells
Summary
Receptor for advanced glycation end-products (RAGE) is one of the members of the immunoglobulin superfamily and a multiligand cell surface receptor with structural homology to other immunoglobulin-like receptors.[1]. All these ligands interact with the extracellular domain of RAGE and activate downstream signaling pathways, including activation of extracellular regulated protein kinases (ERK 1/2) and phosphatidylinositol 3-kinase (PI3K).[9,10,11,12,13] Activated RAGE promotes the production of reactive oxygen species and proinflammatory cytokines, and it inhibits the expression of tumor suppressor genes.[14,15] Overactivation of RAGE is associated with many diseases, including diabetes complications, atherosclerosis, neurodegenerative diseases, liver fibrosis, and tumors.[9,16,17,18,19,20]
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