Fig 2: Model depicting cellular outcomes in esco2 mutant zebrafish embryos and hypothesized impact that compensation might have on RBS phenotypes. (A) A model of the molecular and cellular event ongoing in the esco2 mutant zebrafish. The majority of cells undergo a prolonged mitotic delay ultimately resulting in a single chromatid mis-segregation yielding a micronuclei. We observed: (i) through live imaging an interphase cell with a micronuclei undergo apoptosis, (ii) increased levels of ?-H2ax, indicating a DNA damage response (DDR), and (iii) p53-dependent apoptosis within the neural tube. Together, these data suggest that the micronuclei induce a DDR response to activate the p53 apoptotic response. There is subsequent apoptosis even in the absence of p53, suggesting that an alternative apoptotic signal exists. Other cells within the esco2 mutant embryos appear to undergo a normal mitotic progression, and have only mild cohesion loss, suggesting that compensation mechanisms exist. (B) Depicts a model in which variation in compensation between individuals and between tissues in an individual might explain the phenotypic differences between individuals with RBS.

Fig 3: aTAT1 downregulation increased ?-H2AX but not p-CHK1 or p-CHK2. (a) ?-H2AX level determined by western blotting. (b) Confocal images of HeLa cells immunostained with anti-?-H2AX antibody. (c) HeLa control or aTAT1 stably expressing cells were analyzed by western blotting after sh #1 or sh #2 treatment using antibodies against Ac-Tu, ?-H2AX, pSer345-CHK1, and pThr68-CHK2. Control cells exposed to UV irradiation then recovered for 4 h were used as a positive control. Scale bar, 25 µm.

Fig 4: Depletion of ?113p53 has little effect on DNA damage response and apoptotic activity in zebrafish telencephalons.A–F Cryosections of ?113p53+/+ (A, D) and ?113p53M/M telencephalons (B, E) at 16- or 24-months old were stained with an anti-?-H2AX (in red) antibody. G–L TUNEL assay (in red) was performed on cryosections of ?113p53+/+ (G, J) and ?113p53M/M zebrafish telencephalons (H, K) at 16- and 24-months old, as indicated. The nuclei were stained with DAPI (in blue). An average number of ?-H2AX + cells or apoptotic cells per 100 µm of the ventricular zone (VZ) in one section from the middle region of each telencephalon was presented in C (16-months old) and F (24-months old) or I (16-months old) and L (24-months old), respectively. Framed areas in A, B, D, E, G, H, J, K were magnified in A’, B’, D’, E’, G’, H’, J’, K’, respectively. Scale bar in A, B, D, E, G, H, J, K, 50 µm; Scale bar in A’, B’, D’, E’, G’, H’, J’, K’, 10 µm. Each dot represents the average number of ?-H2AX + cells or apoptotic cells per 100 µm of VZ in one section. About four to five sections were chosen from the middle region of each telencephalon, and at least five telencephalons were sampled in each group. Statistical analysis was performed on relevant data using the Student’s two-tailed t test. N.S., P > 0.05.

Fig 5: Inhibition of YAP nuclear localisation by dasatinib sensitises OVCAR-8 cells to AZD1775-based therapy. (A) The cells were treated with 10 nm dasatinib for 6 h and were stained with the anti-YAP/TAZ antibody. The bar represents 30 µm. Arrowheads indicate nuclear exclusion of YAP/TAZ. (B) The cells were treated with the indicated concentration of AZD1775 with gemcitabine in the presence or absence of 10 nm dasatinib for 4 days. The data are representative of at least three independent experiments. Significant differences between the groups were evaluated using an independent-sample t-test. *P < 0.05. (C) The cells were treated with the indicated concentration of AZD1775 and 1.5 nm gemcitabine for 4 days. The data are representative of at least two independent experiments. cPARP, cleaved PARP. The percentage of cPARP was calculated as in Fig. 2B. (D) The cells were treated with the indicated concentration of AZD1775 with or without 1.5 nm gemcitabine for 4 days. The cells were analysed using flow cytometry. The data are representative of at least two independent experiments. Cells undergoing early and late apoptosis were shown in red and purple rectangles, respectively. (E) The cells were treated with drugs for 2 days. (i) Representative images. (ii) The percentage of ?-H2AX-positive cells was counted. The data represent the mean and standard deviation from three independent experiments. Significant differences between the groups were evaluated using an independent-sample t-test.