RUNX1^R320∗ results in differentiation block and increased DNA damage sensitivity. (A) Representative images of RUNX1 wild-type and RUNX1^R320∗ cells treated with DMSO or 10 nM TPA for 48 hours. Differentiating cells are denoted with arrows. (B-C) Representative flow cytometry analysis of RUNX1 wild-type and RUNX1^R320∗ K562 cells which were treated with DMSO and 5 nM TPA for 48 hours. The MK marker CD61 (integrin β3 chain) was analyzed, along with the erythroid marker CD235a (glycophorin A). Live cells were divided into 4 groups using the FACS diva software based on the presence (+/−) of CD61 and CD235a. DMSO-treated control cells were compared with TPA-treated cells for both the RUNX1 wild-type and RUNX1^R320∗ genotypes (n = 3). Significance was determined using 2-way ANOVA. (D) DNA damage levels in RUNX1 wild-type and RUNX1^R320∗ cells upon treatment with ETOP and CPT relative to the DMSO control. The cells were treated with 25 μM ETOP or CPT for 1 hour at 37°C before fixation and staining. DAPI was used to identify the nuclei of cells and the γH2AX mean signal intensity was measured per cell within the nucleus. Student t test was used to determine significance. ∗ P ≤ .05; ∗∗ P ≤ .01; ∗∗∗ P ≤ .001; ∗∗∗∗ P ≤ .0001. DMSO, dimethyl sulfoxide; FACS, fluorescence-activated cell sorting; DAPI, 4′,6-diamidino-2-phenylindole.