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. 2021 Jun 3;11:11823. doi: 10.1038/s41598-021-90958-1

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Liver injury induces the growth of tumor-initiating cells (TICs) in Sox9-Pten mice concurrent with the activation of Wnt signaling. (A) Accumulation of TIC populations in Sox9-Pten and control mice on DDC vs. NC (n = 3 for each group). TICs characterized by CD133 & CD49f. dual positive (left) or CD44 & CD24 dual positive (right) were significantly increased by DDC in Sox9-Pten mice. The asterisk suggests a significant difference between the two groups at P < 0.05. Liver cells in the control and Sox9-Pten mice treated with DDC or NC were stained for the respective cell surface CD markers followed by quantification using flowcytometer. Data expressed as percentage of dual positive cells over total nonparenchymal cells. Parenchymal cells were excluded during cell isolation. (B) Ki67 staining of liver sections from Sox9-Pten mice treated with DDC or NC. Left, representative image of Ki67 staining (green). Right, quantitation of Ki67 positive cells. The asterisk indicates statistical significance between the indicated groups at P < 0.05. Blue, DAPI. (C) mRNA expression of stem cell markers in control and Sox9-Pten mice treated fed DDC or NC (n = 3–5). Left, Sox9, middle, TROPS2, and right EpCAM. The asterisk indicates statistical significance between the indicated groups at P < 0.05. (D) Immunoblotting analysis of β-catenin and EpCAM in Sox9-Pten and control mice fed on DDC vs. NC. Experiment repeated at least three times with multiple individual mice. (E) Immunofluorescent staining of β-catenin (green) in control and Sox9-Pten livers treated with DDC vs. NC. CK (red) indicate ductal structures. Blue, DAPI.