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. 2020 Oct 28;11(10):925. doi: 10.1038/s41419-020-03126-0

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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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Fig. 4 — A A sphere-forming assay showed that LY CM significantly enhanced the sphere-forming ability, while LY + Met CM attenuated this effect. Bar: 500 μm. Western blot (B), laser confocal microscopy (C), and flow cytometry (D) results showed that the expression of the cancer stem cell markers ALDH1A1, CD44, and Nanog was upregulated in cells stimulated with LY CM, but this upregulation was diminished when cells were treated with LY + Met CM. Bar: 50 μm. E Cells pre-stimulated with CM from different groups were then treated with LY2835219. A CCK8 assay revealed that the cells stimulated with LY CM had a significantly lower inhibition ratio than the control cells, while the cells stimulated with LY + Met CM had an inhibition ratio similar to that of the control cells. F, G An in vivo study demonstrated that the tumorigenic ability of Cal27 cells was upregulated by LY CM but not by LY + Met CM. Tissue immunofluorescence staining (400×) showed that ALDH1A1 and CD44 levels were upregulated in tumors in the LY CM group but not in those in the LY + Met CM group. Bar: 100 μm. H Immunohistochemistry results (400×) for the HSC6 xenograft model showed that ALDH1A1 and CD44 levels were upregulated in tumors in the LY group but not in those in the LY + Met group. Bar: 100 μm. *P < 0.05 when compared with the control group; ^#P < 0.05 when compared with the combined group; ns indicates no significant difference; one-way ANOVA. LY: LY2835219; Met: metformin; CM: conditioned medium.