Increased apoptosis and DNA damage accumulation in stem and myeloid progenitor cells. (A) Annexin V was used to mark apoptotic cells. LSK and MP cells from MDS/MPN hypoallelic mice (backgate analysis shown at left) contain more apoptotic cells (30.5% and 42%, respectively) than age-matched wild-type controls (8.29% and 14.2%, respectively). Percentages shown represent averages of all samples; n = 3 in each group. Both NR4A1^−/−NR4A3^+/− (n = 1, at 11.5 months) and NR4A1^+/−NR4A3^−/− mice (n = 2, at 6-7 months) were used for this assay. *P < .01. Error bars represent SEM. (B) CD11b⁺/Gr-1^low/− cells from MDS/MPN NR4A1^+/−NR4A3^−/− mice (n = 4, at 4-6 months) were analyzed for DNA damage. Bar chart of percentage γH2AX-positive cells in unirradiated samples shows that 3 of 4 hypoallelic mice analyzed have increased basal DNA damage. P = .07 and P = .055 for more than 5 foci and 2 to 5 foci, respectively. Error bars represent SEM. (C-F) CD11b⁺/Gr-1^low/− cells from MDS/MPN hypoallelic mice were analyzed for DNA damage after in vitro exposure to γ-irradiation. (C) Bar chart of percentage of γH2AX positive cells 15 minutes after irradiation shows equal DNA damage induction. (D) Bar chart of percent γH2AX positive cells 3 hours after irradiation. N = 5 in each group. (E-F) At 3 hours after irradiation, anti-γH2AX immunofluorescence (green) showed a significantly higher amount of DNA damage remaining in diseased mice (F) compared to healthy controls (E; 100×/1.30 NA oil objective magnification). Both NR4A1^−/−NR4A3^+/− (n = 2, at 10-12 months) and NR4A1^+/−NR4A3^−/− mice (n = 3, at 5-8 months) were used for this assay. Error bars denote SEM.