Deleting the Runx1 distal isoform does not affect megakaryocyte maturation. (A-D) FACS analysis of BM WT, P1-MRIPV/⁺, and P1-MRIPV/MRIPV megakaryocytes. (A) Representative FACS plots of CD41 and CD42d expression; (B) quantification of CD41⁺ CD42d⁻, and CD41⁺ CD42d⁺ megakaryocytes (N = 6). (C) Representative FACS plots of DNA content (Hoechst 33342 incorporation) of CD41⁺ CD42d⁻ (left) and mature CD41⁺ CD42d⁺ (right) BM megakaryocytes. (D) Mean ploidy (DNA content) of CD41⁺ CD42d⁻, and CD42d⁺ BM megakaryocytes (N = 3). (E-F) Histological analysis of BM megakaryocytes. (E, top) Hematoxylin and eosin (H&E)-stained BM sections. (Bottom) Anti-mouse von Willebrand factor (vWF)-stained BM sections. (F) Enlarged images of megakaryocytes i-ix as indicated in panel E. Representative images from 3 independent experiments are shown. (G-I) CFU-C activity of WT, P1-MRIPV/⁺, and P1-MRIPV/MRIPV PreMegE cells following culture in MegaCult medium. (G) Photographs of representative MegaCult colonies; (H) colony (CFU-C) numbers per 1000 PreMegEs (N = 5); (I) numbers of megakaryocytes per CFU-Mk colony from 3 representative independent experiments (mean ± standard deviation, Mann-Whitney U test). (J-M) Megakaryocyte differentiation in vitro captured by time lapse imaging. (J) Representative images of cytoplasmic extension-forming megakaryocytes (red arrowheads); (K) representative images of a proplatelet-forming megakaryocyte (red arrowheads) at 48 to 68 hours in culture. (L) Quantitation of cytoplasmic extension-forming megakaryocytes from 24 to 120 hours in culture, normalized to the total number of events (visible cells) at 24 hours; (M) quantitation of proplatelet-forming megakaryocytes from 24 to 120 hours normalized to the total number of events at 24 hours (N = 6). *P < .05, **P < .01, ***P < .001, ****P < .0001. Expt, experiment.