Fig 2: DEN-induced liver tumors upregulate caspase-2 expression without a direct effect of DEN on PIDDosome activation Immunoblots of wt non-tumorous (NT) and tumor (T) tissue lysates were probed for CASP2, RAIDD, and p53. HSP90 and PonceauS protein staining served as loading controls.Proliferation of tumor tissue was determined by the percentage of Ki67-positive nuclei analyzed by flow cytometry. T: wt n = 17, Casp2-/- n = 11, Raidd-/- n = 10, Pidd1-/- n = 8; NT: wt n = 12, Casp2-/- n = 11, Raidd-/- n = 7, Pidd1-/- n = 5. N-numbers refer to biological replicates.Experimental outline: 15-day-old (P15) female and male wt and Pidd1-/- mice were injected with DEN (25 mg/kg) or PBS at t(0). The livers were analyzed after t(8 h) and t(48 h), or the mice were weaned at P19 to induce polyploidization and livers harvested 5 days later t(weaning + 5 d). t(8 h) PBS: wt n = 5, Pidd1-/- n = 5; DEN: wt n = 3, Pidd1-/- n = 3. t(48 h) PBS: wt n = 3, Pidd1-/- n = 3; DEN: wt n = 5, Pidd1-/- n = 3. t(weaning + 5 d) PBS: wt n = 3, Pidd1-/- n = 3; DEN: wt n = 3, Pidd1-/- n = 3.Immunoblots of the experiment described in (C) at t(8 h), t(48 h), and t(weaning + 5 d) for CASP2, RAIDD, p53, ?H2AX, and HSP90 expression as loading control.The number of binucleated hepatocytes per field was counted on H&E-stained paraffin sections of PBS treated wt and Pidd1-/- mice at t(0 h) and at t(48 h) after PBS or DEN treatment. N-numbers refer to biological replicates.Immunofluorescence of the same livers shown in (E) performed for the distal centriolar protein CP110 to stain centrioles, ß-catenin for cell borders, and DAPI to stain DNA. Representative images of a mononucleated hepatocyte with 2 centrioles and a binucleated hepatocyte with 4 centrioles are shown.Centriole number was quantified with respect to the number of nuclei per cell in wt and Pidd1-/- livers at the time points described in (E); n = 3 biological replicates. Data information: Data in (B) are represented as median, (E, G) as mean ± SEM, statistical significance was determined by one-way ANOVA with multiplicity correction (Sidak–Holm); *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001. Scale bar size in (F) represents 10 µm. Source data are available online for this figure.

Fig 3: Molecular characterization of PIDDosome-deficient liver cancer specimens Transcript levels of Casp2, Raidd (P = 0.74) and Pidd1 in non-tumorous (NT) and tumor tissue (T) from mice bearing DEN-induced HCC. Hprt mRNA expression was used for normalization.Proliferation was assessed by immunostaining of Ki67 on paraffin-embedded tissue sections; n = 8 per genotype and tissue. Data are shown as median.Transcript levels of Prom1 and Thy1, the genes encoding for the cancer stem cell markers CD133 and CD90, in NT and T of wt and T of Casp2-/- mice with DEN-HCC. The Ct values were normalized over Hprt expression.Representative images of H&E-stained liver sections that were used for the quantification of binucleated cells in Fig 2E (green arrows mark examples). Scale bar represents 100 µm.Transcript levels of Cyp2E1 in wt (n = 8) and Pidd1-/- (n = 5) livers from 15-day-old mice. Data information: Data in (A, C, E) are represented as mean ± SEM from independent biological replicates. Statistical significance was determined by unpaired, two-tailed Student’s t-test or one-way ANOVA with Sidak–Holm correction for multiplicity (C); *P = 0.05, **P = 0.01. N-numbers refer to biological replicates.

Fig 4: Nuclear ploidy and binucleation are reduced in HCC tumor tissue A, B(A) Binucleation was assessed blinded in H&E-stained sections of murine non-tumor (NT) and tumor tissue (T) also shown in (B). T: wt n = 14, Casp2-/- n = 11, Raidd-/- n = 10, Pidd1-/- n = 10; NT: wt n = 8, Casp2-/- n = 10, Raidd-/- n = 8, Pidd1-/- n = 9. N-numbers refer to biological replicates.C, D(C) Representative histograms of nuclei stained for DNA, which were isolated from non-tumor and tumor tissue of wt and Casp2-/- mice. These animals are marked as red “x” in the quantification shown in (D) where the degree of polyploidy is reflected as percentage of octaploid nuclei (T: wt n = 17, Casp2-/- n = 11, Raidd-/- n = 10, Pidd1-/- n = 8; NT: wt n = 12, Casp2-/- n = 11, Raidd-/- n = 7, Pidd1-/- n = 5). N-numbers refer to biological replicates. Data information: Data are represented as median, statistical significance was determined by one-way ANOVA with multiplicity correction (Sidak–Holm); *P = 0.05, **P = 0.01, ***P = 0.001, ****P = 0.0001; scale bar represents 100 µm.

Fig 5: Loss of PIDDosome function reduces tumor formation in DEN-induced HCC Male wt and PIDDosome-deficient mice were injected with 25 mg/kg DEN at the age of 15 days, and their livers were analyzed 10 months later.Numbers of surface tumors per liver were counted.The cumulative diameter of all surface tumors per liver per mouse reflects the overall tumor load.Weight of the livers including tumor mass relative to the body weight (LW/BW ratio) was recorded.Representative pictures of total livers with tumors of the indicated genotypes. Data information: Open symbols in graphs (B–D) represent the mice shown in (E). wt n = 22, Casp2-/- n = 11, Raidd-/- n = 14, Pidd1-/- n = 15. The line in (B–D) represents the median value of each group, and statistical significance was determined by one-way ANOVA with multiplicity correction (Sidak–Holm) and defined as *P = 0.05, **P = 0.01, ***P = 0.001; scale bar in (E) represents 1 cm.