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. 2019 Jul 5;9:9763. doi: 10.1038/s41598-019-45100-7

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© The Author(s) 2019

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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Forced Expression of Pax6 Upregulates Expression of Neural Crest Markers in P10 LNC. Transfection of Ad-GFP (GFP) or Ad-GFP-Pax6 (GFP-Pax6) plasmid (A) was performed in P10 LNC cultured on coated Matrigel in MESCM after their respective multiplicity of infection (MOI) was pre-determined during a period of 5 days (B, *p < 0.1, **p < 0.05, n = 3). Following the respective transfection, RT-PCR analysis was used to compare the transcript levels of ESC markers (Oct4, Sox2, and Nanog) and neural crest markers (p75^NTR, Musashi-1, Nestin, Msx1, and FoxD3) (C, **p < 0.05, n = 3). Western blot analysis was used to compare the protein expression of 46 kDa Pax6, Oct4, p75^NTR, and Musashi-1 using β-actin as the loading control (D). Cytolocalization of Pax6 (red) and Oct4 (green), Pax6 (red) and Sox2 (green), as well as p75^NTR (red) and Musashi-1 (red) (E) were determined by either double or single immunofluorescence staining. Nuclear counterstaining by Hoechst 33342. Scale bar = 100 µm.