(A) Overview of experimental approach: WT TP53 embryos were acquired by breeding WT Tp53 male and WT Tp53 female mice. Tp53-KO embryos were acquired from breeding of Tp53-KO homozygotic males and Tp53-KO heterozygotic females. The E13.5 embryos were obtained from the above breeding, and MEFs were later isolated from these embryos, with Tp53^+/+ and Tp53^–/– genetic backgrounds, respectively. HSPCs were obtained from a healthy donor and isolated to obtain CD34⁺ cells. Onecut3 expression was enforced by the Retro-X Tet-One system in the MEFs, and by ssODN in the human CD34⁺ HSPCs. Then we assessed the morphology, karyotype, and indirect IF. (B) Cell lysates were harvested after treatment with or without Dox (100 ng/mL) in both WT Tp53 and Tp53-KO MEFs for 48 hours and were blotted against anti-ONECUT3 antibodies. Actin was used as a loading control. (C) Representative images of WT Tp53 and Tp53-KO MEFs after 4 days of Dox treatment, followed cytospin and then Wright-Giemsa staining. Graph shows a comparative analysis of the multinucleated cell percentage (n = 3). (D) Chromosome number analysis in over 30 mitotic cells. Data points represent individual WT Tp53 and Tp53-KO MEFs. Conventional MEF chromosome numbers (n = 40 or 80; in grayscale) were considered as a benchmark; deviations were deemed anomalous. (E) Cells were either subjected to induction of ONECUT3 OE (upper panel) or to treatment with 380 μM 5-FU to induce Tp53 expression or with DMSO for 24 hours (lower panel). (F) Analysis of morphology (left) and karyotype (right) for ssODN-mediated control and ONECUT3 expression in human CD34⁺ HSPCs. Red arrows indicate multinucleated cells. Scale bars: 100 μm. Error bars represent the SD. NS, not significant; *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed, paired Student’s t test (C and F–H) or χ² test (D).