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. 2017 Sep 8;7:10941. doi: 10.1038/s41598-017-11287-w

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© The Author(s) 2017

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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Nrp1 expression is important for MaSC colony formation in vitro. (A) qPCR analysis validating the Nrp1 knockdown efficiency of two independent shRNAs. Data are pooled from three independent experiments, are presented as mean ± SEM. ***P < 0.001. (B) Representative images of colonies upon infection with scramble or Nrp1 shRNAs (sh-1, sh-2) viruses (top panels), and quantification of colony sizes at cultured day 6 (bottom panels). Each dot represents one colony. Scale bar, 20 μm. Data are presented as mean ± SEM. ***P < 0.001. (C) Representative images of EdU labeled basal colonies (top panels). EdU⁺ cell percentages (EdU⁺ cells/DAPI⁺ cells in each colony) were quantified at day 6 (bottom panel). Each dot represents one colony. Scale bar, 20μm. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01. (D) Representative images of TUNEL staining of basal colonies (top panels). TUNEL⁺ cell percentages (TUNEL⁺ cells/DAPI⁺ cells in each colony) were quantified at day 6 (bottom panels). Each dot represents one colony. Scale bar, 20μm. Data are presented as mean ± SEM. (E) Serial passage of basal colonies after lentiviral infection with scramble or Nrp1 shRNAs. The colony numbers of each passage were quantified.