There are minimal overlaps in altered genes between DEN-treated wild type and <i>bid</i>-deficient mice, and between mice treated with DEN for different times.

<p>(A-B). Genes with altered expression was compared between the different time points (4–6 month vs 10–12 month) for the DEN-treated wild type (A) and <i>bid</i>-deficient (B) livers. Overlapped genes between the time points were shown in the diagram and listed. (C-D). Genes with altered expression were compared between DEN-treated wild type (WT) and <i>bid</i>-deficient (KO) livers at the time point of 4–6 month (C) or 10–12 month (D). Overlapped genes between the two groups of mice were shown in the diagram and listed. All genes are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155211#pone.0155211.s001" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155211#pone.0155211.s008" target="_blank">S8</a> Tables. Size of the diagram and overlapped region are not proportional to the number of genes.</p

Bid-deficient livers have a lower tumor burden and fewer gene expression alterations following long-term DEN treatment.

<p>Wild type (WT) and <i>bid</i>-deficient (KO) mice were treated with or without DEN for 4–6 or 10–12 months. (<b>A</b>) The percentage of liver weight in the body weight was calculated as a parameter for tumor burden. The numbers (n) of mice in each group were shown. (B). The livers were subjected to microarray analysis. The data in each group were averaged and the numbers of genes with increased and decreased expression in the DEN-treated livers in comparison with the normal age-matched samples were shown in solid and open columns, respectively.</p

Biological pathways related to metabolism and diseases are most affected by DEN-induced carcinogenesis.

<p>GSEA-identified biological pathways were grouped into six functional groups per KEGG pathway analysis for wild type (WT) and <i>bid</i>-deficient (KO) mice given DEN for 4–6 months or 10–12 months. The number of downregulated pathways (A-D) and the upregulated pathways (E-H) were compared among different mouse groups. The most commonly downregulated biological pathway cluster was related to metabolism (cluster 5, A-D), whereas the most commonly upregulated biology pathway cluster was related to human diseases (cluster 4, E-H). For these two clusters, wild type mice had more pathways affected than <i>bid</i>-deficient mice; and later stage (10–12 months) group had more pathways affected than the early stage (4–6 months) group. Pathway functional groups: 1: Cellular Process; 2: Environmental Information Processing; 3: Genetic Information Processing; 4: Human Diseases; 5: Metabolism; 6: Organismal Systems.</p

Bid promotes DEN-induced hepatic carcinogenesis.

<p>The BH3-only Bcl-2 family molecule Bid can promote DEN-induced hepatic carcinogenesis by enhancing cell proliferation. This function may be mediated by at least two mechanisms. One is internally stimulated by oncogenic transformation, which can be regulated by Bid’s action on calcium release from ER, which is required for cell cycle entrance. The other is externally stimulated by cell death, which could be also affected by Bid-mediated pathway. Although the mechanism is not clear DEN-induced liver injury could be affected by Bid. There could be necrotic cell death, either secondary to apoptosis, or primarily due to DEN-induced damage. Immune response and/or inflammation responses are then activated. Bid can participate in inflammasome activation caused by mitochondrial damage [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155211#pone.0155211.ref010" target="_blank">10</a>]. These factors can stimulate proliferation of neighboring cells that are mutated but survived. This process is known as compensatory proliferation. Note that Bid may at the same time regulate the proliferation of these survived neighboring cells through the intrinsic mechanism on ER-calcium release. Tumor cells may become dedifferentiated and lose some of the key hepatic metabolic functions, such as that for amino acids. Gene expression data in this study indicate a significant upregulation of genes involved in cancer growth and in immune/inflammation response, and a significant downregulation of genes involved in metabolism. All of these changes are diminished in the absence of Bid, suggesting the role of Bid in regulating these events.</p

Confirmation of gene expression by qRT-PCR.

<p>Total RNA was extracted from the livers of wild type and <i>bid</i>-deficient mice treated with or without DEN for 10–12 months. qRT-PCR was performed with primers for the indicated genes that were elevated (A), or suppressed (B) in expression in wild type (WT) mice treated with DEN for 10–12 month. Expressions in <i>bid</i>-deficient mice (KO) were also assessed but did not show significant changes for these genes. The fold of changes over the non-treated controls was calculated. Mean+/-SEM (n = 3–4 per group).</p

Significantly more biological pathways are altered in wild type mice than in <i>bid</i>-deficient mice following DEN treatment.

<p>(A). Genes showing significantly altered expressions in DEN-treated wild type (WT) and <i>bid</i>-deficient (KO) liver were subjected to DAVID analysis coupled with KEGG Pathway analysis. (B). All genes in DEN-treated wild type (WT) and <i>bid</i>-deficient (KO) liver were subjected to GSEA coupled with KEGG Pathway analysis. The number of upregulated (increase) or downregulated (decrease) pathways in each group was plotted and indicated. Wild type mice treated with DEN for 10–12 months had the largest number of pathways affected.</p

DEN treatment affects different functional pathways to different degrees in wild type vs <i>bid</i>-deficient mice.

<p>GSEA-identified biological pathways were grouped into six functional groups per KEGG pathway analysis for wild type (WT) and <i>bid</i>-deficient (KO) mice given DEN for 4–6 months or 10–12 months. Upregulated (increased, inner circle) and downregulated (decreased, outer circle) functional groups were displayed based on the percentage distribution. Pathways related to metabolism and diseases are most frequently affected. Functional groups that were not detected were not shown in the diagram.</p