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. 2018 Aug 8;9:3164. doi: 10.1038/s41467-018-05550-5

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© The Author(s) 2018

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. 3 — Alb-R26^Met tumours are characterised by an enrichment in genes overexpressed and with hypermethylated CGIs. a Histogram reporting the KEGG pathway enrichment analysis for genes with changes in CGI methylation and expression in Alb-R26^Met tumours, ordered according to the −Log₁₀ P-value. b Left: Methylation differences versus expression for all genes with CGIs hypermethylated (H⁺) or hypomethylated (H⁻) in Alb-R26^Met tumours. Expression values are relative to controls. Dots correspond to single differentially methylated CpG and the corresponding gene expression (genes which expression is significantly below or above Log₂FC ± 1 are indicated in red). Right: Graph reporting the percentage of downregulated (E⁻) and upregulated (E⁺) genes among those with a hypomethylated (H⁻) or hypermethylated (H⁺) CGI. Note the enrichment of genes overexpressed and with hypermethylated CGIs (indicated by an arrow), on which subsequent studies were focused. c For the 55 genes overexpressed and with hypermethylated CGI in Alb-R26^Met tumours, 3D density plot shows their distribution according to relative position to the ATG (as percentage), gene expression level (as Log₂FC) and CGI methylation (as β-value difference). Note that genes segregate into two groups, according to their relative position to the ATG. d Graph reporting the individual expression level (as Log₂FC; from RNA-seq data) of hypermethylated and overexpressed genes found in Alb-R26^Met tumours compare to controls. Note that the relative position of the hypermethylated CGI to the ATG well segregate the two groups (indicated with a red arrow). e Box plot illustrating the global expression levels of genes in Group-I and Group-II. f Graph reporting individual expression levels (as Log₂FC; by RT-qPCR) of genes belonging to the two groups in Alb-R26^Met tumours (n = 8) relative to control livers (n = 6). Red lines report the median Log₂FC in expression. Note that genes are distributed according to the location of the hypermethylated CGI relative to the ATG. g Box plot showing the global Log₂FC in expression (according to data in F) of genes in Group-I and Group-II. In e and g, the median is reported by a line and bars extend to the minimum/maximum values. Significance is indicated on the top. *P < 0.05, **P < 0.01, ***P < 0.001