Fig 2: BRCA1mut-iPSC-derived FTE organoids reveal ovarian cancer progression of carcinogenesis in vitro(A) H&E images for control (80i-CTR) and BRCA1mut-FTE organoids (79i-BRCA) at 1 year in culture.(B) Immunocytochemistry of FTE markers OVGP1 and TUBB4A in human FTE tissue and control and BRCA1mut-FTE organoids. MUC16 and TP53 are shown as markers for neoplastic transformation in control and BRCA1mut-FTE organoids at 1 year in culture. Scale bars, 50 µm. Percentage of TP53 was calculated for n = 3 different controls and n = 3 different BRCA1mut-FTE organoids.(C) Expression of BRCA1, ATM, ATR, and 53BP1 genes at 4 months and 1 year in culture in control and BRCA1mut-FTE organoids. Significance was calculated using Student’s t test; *p < 0.05.(D) Differential gene expression and GSEA for control and BRCA1mut-FTE organoids at 1 year in culture. FDR value is <0.05. Z scores of the leading-edge genes were calculated and used to construct a heatmap.

Fig 3: Characterization of A549 WT and A549 TP53 KO isogenic cell pair. A, Cell lines were treated with a 6 Gy dose of radiotherapy and analyzed at the times indicated. Cell extracts were western blotted for total and phosphorylated (p-) proteins. Vinculin served as a total loading control. B, Clonogenic survival of cell lines after exposure to increasing doses of radiotherapy. C, Clonogenic survival (normalized to untreated DMSO controls of the respective radiotherapy treatment) of cell lines in response to concentrations indicated for AZD0156 alone (black line; 0 Gy) or after radiotherapy at the doses indicated (red line). D, Approximate radiotherapy DEFs at 75% (aDEF75) of each concentration of AZD0156 as determined in (C), with overall dose enhancement determined as significantly greater in TP53 KO cells. Graphs in B–D represent the medians of two independent experiments performed in triplicate, with error bars representing upper and lower limits. Statistical significance was tested using a two-way ANOVA and is indicated where *, P < 0.05; **, P < 0.01; or ***, P < 0.001.

Fig 4: Heterochromatin primarily suppresses the p53-mediated apoptosis pathway. OS was generated by treatment with 10 mU/mL GO for 3 h in the indicated assays. (A) KEGG enrichment analysis of genes with significant expression changes in satellite a- vs. GFP-transfected ARPE-19 cells (1.5-fold change; P < 0.01). (B) WB analysis of p53Ser15 phosphorylation in GFP-, satellite a-, or major satellite-transfected ARPE-19. (C) qRT-PCR analysis of p53-regulated apoptotic genes in ARPE-19 transfected with indicated plasmids with or without OS exposure. **P < 0.005; *P < 0.05. (D) WB analysis of p53ser15 phosphorylation in ARPE-19 treated with indicated concentrations of chaetocin for 24 h. (E) qRT-PCR analysis of p53-regulated genes in ARPE-19 with or without 500 nM chaetocin treatment for 24 h before further OS exposure. *P < 0.05; **P < 0.005; ***P < 0.001. (F) ChIP-seq analysis of H3K9me3 signals in ARPE-19. (G and H) qChIP experiments showing increased H3K9me3 and p53 occupancy on p53 target promoters on OS exposure. (I) qChIP experiments showing H3K9me3 occupancy on p53 target gene promoters in control (Ctrl) or p53 knockdown (p53si) hRPE cells. p53 knockdown efficiency is shown in Fig. S8. siRNA#1 was selected in ChIP experiments. Error bars represent SD (n = 2). *P < 0.05; **P < 0.01. (J) qRT-PCR analysis of p53-regulated transcripts in HCT116 p53+/+ and HCT116 p53-/- cells transfected with indicated plasmids. n = 3. *P < 0.05; **P < 0.01.

Fig 5: DDX11 helicase activity supports cohesion, DNA replication and embryonic survival.a WABS01 cells (left panel) and RPE1-TP53-DDX11KO cells (right panel) were stably transduced with empty vector (EV), wtDDX11 or DDX11-K50R and analyzed by Western blot. l.e. long exposure. A representative of two independent protein analyses is shown. b Cells were analyzed for cohesion defects. Per condition, in total 100 metaphases from two independent experiments were assessed. c Replication fork speed was assessed with a DNA fiber assay using a double labeling protocol. In total at least 200 fibers were scored per condition in two independent experiments. The example track represents an ongoing fork. Blue dots, DDX11 proficient; red dots, DDX11 deficient. Black lines indicate the mean. P-values were calculated by a non-parametric one-way ANOVA test. d WABS01 cells, stably transfected with different DDX11 mutants, were analyzed for cohesion defects. Per condition, in total 150 metaphases from three independent experiments were assessed. e Alignment of different DDX11 orthologues (top) and related SF2 DNA helicases (bottom). Conserved motifs are indicated. Arrows point at the positions of K50R, G57R, and C705Y mutations. f In vitro DNA helicase assay of patient-derived DDX11 mutants. Purified, recombinant Flag-tagged proteins were analyzed by western blot (left). Proteins were incubated at increasing concentrations with a fluorescently labeled forked DNA substrate. Helicase reaction products were resolved by gel electrophoresis. Percentage of displaced DNA was determined using Image-J software. Right panel: quantification from three independent experiments. g DDX11-G57R heterozygous mice were generated by oligonucleotide-directed gene modification in mouse embryonic stem cells substituting glycine codon 57 for an arginine codon (C>G substitution). Subsequent intercrossing of heterozygous mice failed to produce homozygous offspring.