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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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Inducing liver injury with DDC accelerates liver carcinogenesis in Sox9-Pten mice. (A) Protocol for tamoxifen injection and DDC treatment. Tamoxifen injected Sox9-Pten (n = 5) and control (n = 6) mice were fed on DDC for 3 months to induce liver injury before liver tissues were collected for analysis at 6 months old. Normal chow (NC) was used as diet control for DDC treatment in Sox9-Pten (n = 8) and control (n = 6) mice, respectively. (B) Representative microscopic images of the livers from Sox9-Pten and control mice on DDC and NC. a, Pten genotype control mice fed on NC. b. Sox9-Pten mice fed on NC. c. Pten genotype control mic fed on DDC diet. d. Sox9-Pten mice fed on DDC diet. (C) Quantitative analysis of tumors development in Sox9-Pten mice and control mice treated with DDC vs. NC. (D) Immunochemical and H&E staining on serial sections of the tumors developed in DDC treated Sox9-Pten mice vs. controls. Panels a&b, Control mice treated with DDC. Panels c & d, Sox9-Pten mice treated with DDC. Green, YFP. Red, either HNF4α (a and a′; c and c′) or CK (b and b′; d and d′). Both ductal hyperplasia in control (b and b′) and tumor formation in Sox9-Pten mice (d and d′) are positive for YFP, indicating that these are SOX9⁺ cells in origin. Panels a′-d′, amplified views of the boxed areas in a-d respectively. Blue, DAPI. Control mice are: Pten ^loxP/loxP; Cre^- mice with no treatment; Pten^+/+; Sox9-CreERT mice treated with either corn oil (CO) or tamoxifen; or Pten^loxP/loxP; Sox9-CreERT mice treated with coil oil.

Inducing liver injury with DDC accelerates liver carcinogenesis in Sox9-Pten mice. (A) Protocol for tamoxifen injection and DDC treatment. Tamoxifen injected Sox9-Pten (n = 5) and control (n = 6) mice were fed on DDC for 3 months to induce liver injury before liver tissues were collected for analysis at 6 months old. Normal chow (NC) was used as diet control for DDC treatment in Sox9-Pten (n = 8) and control (n = 6) mice, respectively. (B) Representative microscopic images of the livers from Sox9-Pten and control mice on DDC and NC. a, Pten genotype control mice fed on NC. b. Sox9-Pten mice fed on NC. c. Pten genotype control mic fed on DDC diet. d. Sox9-Pten mice fed on DDC diet. (C) Quantitative analysis of tumors development in Sox9-Pten mice and control mice treated with DDC vs. NC. (D) Immunochemical and H&E staining on serial sections of the tumors developed in DDC treated Sox9-Pten mice vs. controls. Panels a&b, Control mice treated with DDC. Panels c & d, Sox9-Pten mice treated with DDC. Green, YFP. Red, either HNF4α (a and a′; c and c′) or CK (b and b′; d and d′). Both ductal hyperplasia in control (b and b′) and tumor formation in Sox9-Pten mice (d and d′) are positive for YFP, indicating that these are SOX9⁺ cells in origin. Panels a′-d′, amplified views of the boxed areas in a-d respectively. Blue, DAPI. Control mice are: Pten ^loxP/loxP; Cre^- mice with no treatment; Pten^+/+; Sox9-CreERT mice treated with either corn oil (CO) or tamoxifen; or Pten^loxP/loxP; Sox9-CreERT mice treated with coil oil.