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| Generation of Two Mouse Melanoma Cell Lines Stable Overexpression or Silencing of Nodal and Identification of EMT Phenotype |
| QUAN Mei-yu, GUO Qiang, ZHANG Kun-shui, FANF Rui, LI Cui-lin, DU Jun |
| Department of Microbial and Biochemical Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China |
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Abstract Objective: To generate two B16 melanoma cell lines, stably overexpress or silence Nodal gene and identified their EMT markers for study of phenotypic changes and molecular mechanisms of Nodal induced EMT in melanoma. Methods: The mouse melanoma B16 cells were transfected with recombinant plasmid pL-tdTomato-mNodal or shRNA plasmid pGFP-V-RS-Nodal respectively. The stable cell lines B16/dT-mNodal and B16/sh-Nodal were obtained after resistance screening and enrichment, positive clone selection and expasion. The intracellular gene and protein levels of Nodal and EMT markers in these two cell lines were detected by qRT-PCR and Western blotting. Results: Both stable cell lines were constructed successfully. B16/dT-mNoda cell line exhibited strong red fluorescence with obvious up-regulated of Nodal mRNA and protein and Showed the characteristics of mesenchymal cell. While B16/sh-Nodal cell line displayed strong green fluorescence, as well as notable down-regulated Nodal mRNA and protein levels and Showed the characteristics of epithelial cell. Conclusion: The Nodal stably overexpressing or silencing cell lines B16/dT-mNodal and B16/sh-Nodal have been constructed successfully, providing important experimental tools for the study of Nodal function in the process of melanoma EMT.
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Received: 24 October 2013
Published: 25 March 2014
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