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. 2020 Feb 6;10:1971. doi: 10.1038/s41598-020-58996-3

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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The NME1^LOW subpopulation exhibits a gene expression profile consistent with a neural crest-like phenotype. (a) Isolation of NME1^LOW and NME1^HIGH subpopulations from WM9 clone 21 by FACS for RNA-seq analysis. (b) Volcano plot of mRNA expression in NME1^LOW vs. NME1^HIGH subpopulations. (c) Flow chart of steps involved in identification of two GO biological processes that were upregulated in the NME1^LOW subpopulation. (d) Processes associated with the biological process anatomical structure/morphogenesis. Processes identified as “Hallmarks of Cancer” or associated with “Development” are highlighted with red or blue asterisks, respectively. (e) Validation of RNA-seq analysis across multiple NME1-EGFP-expressing clones. Steady-state expression of mRNAs encoding NME1, GFRA1, AKT3 and SLC14a2 was determined by qRT-PCR in the indicated parental clones (“P”) and NME1^LOW (“L”) subpopulations. Asterisks denote significant differences between matched parental clone and NME1^LOW (“L”) subpopulations, as determined by paired t-test (p < 0.05) (f) Inductions in expression of RNAs encoding EPHAA4 and GFRA1 are reflected in expression of the cognate cell surface proteins at the cell surface (p < 0.05, t-test).