Cytokinesis failure results in DNA damage in RhoA^Δ/Δ fetal liver erythroblasts, as evidenced by staining for the DNA damage marker γH2AX. (A) Fetal liver cells from E14.0 WT and RhoA^Δ/Δ embryos were analyzed by imaging flow cytometry as shown before,¹⁸  up to the gating of erythroblasts (Ter119⁺DAPI⁺ cells). Further analysis based on the size of the nucleus (area_DAPI) and the shape of the nucleus (aspect ratio_DAPI=ratio of the minor axis/major axis) gave a population (gated by the pink line) enriched in binucleated cells. Truly binucleated or multinucleated cells were then identified by visual observation and marked blue in the dot plot. (B-C) Concurrent staining for the DNA damage marker γH2AX revealed that γH2AX, as measured in arbitrary units of fluorescence, was significantly higher in the RhoA^Δ/Δ erythroblasts compared with WT. Considering a threshold for H2AX positivity at 1.5 × 10⁵, the percentage of γH2AX-positive cells was 18.5 ± 4.8% for RhoA^∆/∆ Ter119⁺ nucleated fetal liver cells vs 3.1 ± 1.3% for the WT counterparts (B). The difference was more significant in the multinucleated erythroblasts: 39 ± 13.7% in RhoA^Δ/Δ vs 2.3 ± 1.4% in the WT (C). Mean and median values of the fluorescence intensity of γH2AX in the WT and RhoA^Δ/Δ erythroid precursors are shown (P < .0001 of RhoA^Δ/Δ vs WT). At least 5000 Ter119⁺;DAPI⁺ cells were analyzed in each experiment and results are representative of 3 biological repeats for each genotype. (D) Binucleated RhoA^Δ/Δ cells were strongly positive for γH2AX, indicative of DNA damage. In contrast, binucleated WT cells were negative for γH2AX and likely normal mitotic cells. Three representative cells are shown for each genotype of at least 250 RhoA^Δ/Δ and 40 WT hyperdiploid cells with similar morphology. Images were obtained with a 60× objective lens by ImageStream^X. DAPI was used as a nuclear stain. (E) Fetal liver cells from E14 embryos were stained for γH2AX foci and imaged by confocal microscopy. Cells with >4 foci per nucleus were counted as positive, and at least 50 cells were evaluated per sample. The percentage of γH2AX-positive cells was 38.5% for RhoA^∆/∆ vs 2% for the WT. Bottom panels show the nuclear stain merged with the corresponding brightfield image to demonstrate the binucleated and dysplastic RhoA^∆/∆ cells. The scale bar represents 10 µm.