Fig 2: Spontaneous chromosomal instability in Fancc-/-; Mad2+/- mice. (A) Graphic illustrating the rationale of the quantitative red blood cell (RBC) micronucleation flow cytometry assay. Unlike normal nuclei, micronuclei (MN) arising from mitotic chromosome mis-segregation are not efficiently removed from maturing erythroblasts and therefore, DNA-positive mature RBCs signify abnormal chromosome sorting during erythropoiesis. (B) Example of a micronucleated cell in Fancc-/-; Mad2+/- bone marrow. Red arrows point to micronuclei (MNs). Nuc, nucleus. (C, D) Flow cytometry demonstrating an increased frequency of chromosome mis-segregation (p<0.0001) during Fancc-/-; Mad2+/- erythropoiesis compared to all other mouse genotypes. CD71 was used as a marker for immature peripheral blood red blood cells (RBCs). Frequencies of DNA-containing mature RBCs (CD71-, propidium iodide+) were statistically compared using Fisher’s exact test with n of at least 9 age-matched mice per genotype. (E, F) Karyotype demonstrating genomic instability in non-malignant Fancc-/-;Mad2+/- hematopoietic cells. Normal murine chromosome complement is 40, thus the presence of cells with <40 or >40 chromosomes is suggestive of aneuploidy. Statistical analysis was performed using Fisher’s exact test and Bonferroni post-hoc analysis (n=150 metaphase spreads per genotype, ****p < 0.0001, **p = 0.001).

Fig 3: SUN2 depletion causes metaphase delay and mitotic spindle defects. (A) HeLa Kyoto EGFP-LaminA/H2B-mCherry cells cultured on glass were depleted for SUN2 and imaged using live confocal microscopy. The duration of of each mitosis stage was calculated using the NEBD, shown by lamin A staining (green), as the starting point. The diagram represents the method used to calculate the phases of mitosis. (Scale bars, 20 µm.) (B) Corresponding quantifications of mitotic phase duration: prometaphase (PM), metaphase (M), and anaphase (A) (n = 101 cells for control siRNA and n = 107 cells for SUN2 siRNA from three independent experiments; error bars indicate means ± SEM; ns, not significant; ****P < 0.0001, two-way ANOVA, Sidak’s multiple-comparisons posttest). (C) HeLa Kyoto EGFP-a-tubulin/H2B-mCherry cells cultured on glass were depleted for SUN2 and imaged using live confocal microscopy, and mean mitotic spindle length was calculated from the onset of metaphase to the image just before the onset of anaphase. The diagram represents the method used to calculate mitotic spindle length. (Scale bars, 10 µm.) (D) Corresponding quantifications of mean mitotic spindle length were calculated over metaphase duration (n = 42 cells for control siRNA and n = 56 cells for SUN2 siRNA from three independent experiments; error bars indicate means ± SEM; ***P < 0.001, unpaired t test). (E) HeLa Kyoto Mad2-LAP/H2B-mCherry cells cultured on glass were depleted for SUN2 and immunostained for centromeres (CREST). Representative images show the stainings of histone H2B (blue), CREST (green), and MAD2 (red). The white arrowheads show MAD2 staining. (Scale bars, 10 µm.) (F) Corresponding quantifications of the percentage of cells with MAD2-positive kinetochores (n = 50 cells for control siRNA and n = 49 cells for SUN2 siRNA from two independent experiments; error bars indicate means ± SEM; *P < 0.05, **P < 0.01, two-way ANOVA, Sidak’s multiple-comparisons posttest). (G) HeLa cells cultured on plastic were transfected either with control, SUN2, MAD2, or both SUN2 and MAD2 siRNAs. Mitosis duration was calculated using phase-contrast microscopy as the elapsed time between the onset of rounding and the first image demonstrating cytokinesis and postmitotic spreading (n = 100 cells for each condition from two independent experiments; error bars indicate means ± SEM; ns, not significant; ****P < 0.0001, one-way ANOVA, Tukey’s multiple-comparisons posttest). (H) HeLa cells cultured on glass were fixed and immunostained for SUN2 (magenta) and pericentrin (green). DNA was stained with DAPI (blue). Pictures of cells in metaphase were taken. (Scale bar, 10 µm.) (I) Raw fluorescence intensity profiles of SUN2 and pericentrin were plotted along the line drawn between the centrosomes in metaphase (1). The mean SUN2 fluorescence intensity was measured in the following areas delimited by pericentrin fluorescence intensity: peripheral (P1 and P2), at the centrosome (C1 and C2), and interpolar (I). (J) Representative fluorescence intensity profile of SUN2 and pericentrin in metaphase. AU, arbitrary units. (K) Mean fluorescence intensity of SUN2 and pericentrin in metaphase in the areas delimited by pericentrin staining (n = 44 cells from two independent experiments; error bars indicate means ± SEM; ****P < 0.0001, ***P < 0.001, **P < 0.01, Kruskal–Wallis test, Dunn’s multiple-comparisons posttest performed for SUN2 mean fluorescence intensity).

Fig 4: Knockdown of SETDB1 impaired cell proliferation and cell cycle transition. (A) Representative images of EdU incorporation in siSETDB1 and siCtrl cells. The proliferative cells were detected by Cell-Light EdU Apollo488 (green), and the nucleus was labelled with DAPI (blue). (B) Quantification of EdU incorporation in siSETDB1 and siCtrl cells. (C) Cell cycle analysis of siSETDB1 and siCtrl cells using flow cytometry. Proportion of the G2 stage cells is shown in the column diagram. The blank triangle indicates the peak of the Y-axis. (D) Western blot analysis of the protein levels of MAD2 and ACTB in porcine SSCs. The protein level of SETDB1 relative to ACTB is shown in the column diagram. Significance was presented as ** p < 0.01 or *** p < 0.001. Error bars represented the standard error of the mean (SEM).

Fig 5: Lethal hematopoietic malignancy in wt mice transplanted with marrows of moribund Fancc-/-; Mad2+/- mice. (A) Experimental design of competitive hematopoietic cell transplantation. (B) Disease-free survival (p<0.0001) and (C) overall survival (p=0.043) of wt recipients transplanted with bone marrows of indicated genotypes, n =8 recipients of wt donor marrow and n=13 recipients of marrow from leukemic Fancc-/-; Mad2+/- donors. Statistical significance was calculated via log-rank Kaplan-Meier Mantel-Cox tests. (D–G) Comparison of peripheral blood and marrow flow cytometry, bone marrow and liver morphology in a wt mouse, Fancc-/-; Mad2+/- donor and wt recipient of Fancc-/-; Mad2+/- marrow. (D, E) Myeloid marker flow cytometry, (F) evaluation of bone marrow architecture and (G) liver infiltrates demonstrating similar immunophenotype and histopathologic findings in leukemic donor and recipients. (H) Malignant cell infiltration of lymph nodes and spleen in a representative moribund transplant recipient. (I) Survival of three recipient mice transplanted with bone marrow from a Fancc-/-; Mad2+/- donor mouse (p=0.0246). Statistical significance was determined with log-rank Kaplan-Meier Mantel-Cox tests. *p < 0.05, ****p < 0.0001.