Journal: bioRxiv

Article Title: Tumor Genotype Dictates Mitochondrial and Immune Vulnerabilities in Liver Cancer

doi: 10.1101/2025.09.10.675369

Figure Lengend Snippet: a. Example immunoblots showing enrichment of mitochondrial protein and depletion of other compartments (i.e., cytosol and lysosomes) from healthy liver and tumor mito-immunopurified fractions. p53: c-MYC; p53 -/- , Pten: c-MYC; Pten -/- , Kras: Kras G12D ; p53 -/- . Mitochondrial markers: CS (citrate synthase), VDAC, OGDH, CISD1 and SLC25A12. Cytosolic marker: GAPDH. Lysosomal marker: CTSC (cathepsin C). Input designates whole tumor lysate, MITO-IP is the mito-immunopurified fraction. b. Box plots for example proteins in mitochondrial translation (MRPS23), mitochondrial respiratory chain (UQCC3), iron-sulfur cluster assembly (NFS1), TCA cycle (OGDH), fatty acid oxidation (ECHS1), branched chain amino acid (BCAA) catabolism (BCKDHA), glutamine catabolism (GLS2), urea cycle (CPS1); and their expression changes as median normalized z-scores. Median is indicated as line across the box; minimum and maximum values define upper and lower boundaries c. Heatmap for OXPHOS proteins in c-MYC; p53 -/- and Kras G12D ; p53 -/- models. Rows are clustered by hierarchical clustering. Column min and max are distributed across color scale (log scale). Immunoblot for select OXPHOS components in c-MYC; p53 -/- and Kras G12D ; p53 -/- tumors (three independent tumors of each genotype) shows enrichment in c-MYC -driven tumors. GAPDH was used as control (non-OXPHOS protein). d. PCA plot for mitochondrial proteome of c-MYC -driven PLC models e. IACS-10759 treatment of c-MYC; p53 -/- and Kras G12D ; p53 -/- tumor bearing mice. IVIS images of pre- and post-treatment tumor bearing mice (two representative mice per group) are displayed. Normalized bioluminescence (BL) flux data post-treatment is plotted as bar graph, showing c-MYC -specific tumor suppressive effect of IACS-10759. P -values are calculated by Mann-Whitney U-test with 95% confidence interval (CI). n.s., not significant. f. Heatmap for mitochondrial translation machinery components in c-MYC; p53 -/- and Kras G12D ; p53 -/- models. Columns and rows are clustered by hierarchical clustering. Column min and max are distributed across color scale (log scale). Immunoblot for select mitochondrial translation components in healthy liver tissue, c-MYC; p53 -/- and Kras G12D ; p53 -/- tumors is displayed on the left bottom panel. Middle top panel: Experimental design for generating Mrps22 -depleted c-MYC; p53 -/- tumors. IVIS images of representative mice bearing tumors are shown below. Plot on the right displays quantification for control (sgCtrl) and Mrps22 -depleted (sg Mrps22 ) c-MYC; p53 -/- PLC tumors. P -value is calculated by Mann-Whitney U-test with 95% confidence interval.

Article Snippet: Primary antibodies used are Citrate Synthase (Cell Signaling Technology, 14309S), VDAC (Cell Signaling Technology, 4661S), CISD1 (Cell Signaling Technology, 83775S), OGDH (Proteintech, 15212-1-AP), GAPDH (GeneTex, GTX627408), SLC25A12 (abcam, 200201), Beta-actin (GeneTex, GTX109639), Cathepsin C (CTSC, Santa Cruz Biotechnology sc-74590), MRPS35 (Proteintech, 16457-1-AP), MRPS23 (Proteintech, 18345-1-AP), MRPS22 (Proteintech, 10984-1-AP), MRPL3 (Proteintech, 16584-1-AP), NDUFS1 (Proteintech, 12444-1-AP), NDUFB8 (Cell Signaling Technology, 73951S), Total OXPHOS cocktail (abcam, 110411), GATM (Proteintech, 66322-1-Ig), KRAS (Proteintech, 12063-1-AP), Phospho Acetyl CoA Carboxylase (Ser79) Antibody (Cell Signaling Technology, 3661S), PCYT2 (Proteintech, 14827-1-AP), Beta-tubulin (GeneTex, GTX101279), cleaved caspase-8 (Cell Signaling Technology, 8592T), cleaved caspase-3 (Cell Signaling Technology, 9661T), PTEN (Protein, 22034-1-AP), E-cadherin (Cell Signaling Technology, 14472S).

Techniques: Western Blot, Marker, Expressing, Control, MANN-WHITNEY