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. 2020 Feb 21;10:3156. doi: 10.1038/s41598-020-60145-9

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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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3D cultures of colorectal cancer cell lines LoVo and LS174T. LoVo cells (a–c) or LS174T cells (d–f) were adapted to SCM and used for 3D cultures. (a,d) Growth of the 3D cultures was analysed with CellTiter-Glo. Data represent mean + s.d. (n = 6). (b,e) Representative whole mount immunostaining images of phalloidin (white) and DAPI (blue) in cultures on Day 13. Scale bars = 100 μm. (c,f) Expression of CSC (LGR5, CD44, PROM1) and differentiation (CDX1, CDX2, KRT20) markers by quantitative RT-PCR analysis. With LoVo cells, cultures on Day 10 and 13 were examined for stemCO and diffCO culture conditions, respectively. With LS174T cells, cultures on Day 13 were examined for both culture conditions. Data represent mean + s.d. (n = 3). *p < 0.05, Student t test.