Top protein hits discovered in DEN-induced liver cancer are confirmed in the validation cohort. (a) Dot plots show the distributions of the intensities or mtFE values of the tissue markers verified across control and cancer/DEN samples for discovery cohort (left) and validation cohort (right), respectively. P corresponds to either adjusted p-value from ANOVA analysis (discovery cohort) or p-value from independent paired t-test statistics (validation cohort). All dots in gray represent WT mice, while yellow represents KO mice. (b) ROC curves computed in the validation cohort represent an estimate of predictive protein ability for three respective data matrices depicted by three color codes: lysate (dark red), mito (dark green) and mtFE (dark violet). (c–e) Heat maps for cancer and control samples reflect congruent proteomic changes between the discovery and validation cohorts associated with confirmed biomarkers for lysate (c), mito (d), and mtFE (e) data. P-value of enriched functional categories corresponds to DAVID EASE Score, a modified Fisher Exact P-Value, for gene-enrichment analysis. See also supplementary excel Table S6. (f) Graphical representation of intracellular processes revealed by heat maps of lysate (c), mito (d) and mtFE (e) data in surrounding mitochondria environment in tumor tissue. Conventional expression data measured in mitochondria-enriched fraction after extended DEN-treatment, reflects decrease of ETC Complex I located in the inner membrane. Increased ROS further damages Complex I and affects the release of superoxide60, while increased accumulation of apoptotic modulators in the mitochondrial inner membrane and for redox-sensitive proteins is visible by depletion of SOD2 mtFE scores, which provokes lipid peroxidation (see marker Gsta4)61,62. These processes, reflected exclusively by mtFE markers, can in turn activate the retrograde signaling to cytosol and nucleus48 that influence an increase in ribosomal protein expression reflected by total cellular lysate.