Animals that underwent transplantation with JAK2T875N show increased spleen myeloid colony-forming progenitors and abnormal megakaryocyte ploidy content. Myeloid (CFU-GM, CFU-GEMM, BFU-E) and megakaryocyte colony-forming assays were performed, respectively, in methylcellulose supplemented with IL-3, IL-6, SCF, and EPO and collagen-based media supplemented with TPO, IL-11, IL-3, and IL-6. The experiment was performed in triplicate and the mean plus or minus SD colony number obtained from spleen cells (A) or bone marrow cells (B) is shown. For myeloid colonies, average ratio of BFU-Es, CFU-GEMMs, and CFU-GMs is represented. Of note, BFU-Es were identified in JAK2T875N-derived colonies but not in JAK2WT-derived colonies. WT: JAK2WT; TN: JAK2T875N; Mk: megakaryocyte colonies; BM: bone marrow. Student t test was performed and P value is indicated. For ploidy analysis, bone marrow cells from animals that underwent transplantation were cultured for 4 days in liquid media containing TPO and SCF. Terminal differentiation with proplatelet formation was achieved with both JAK2WT- and JAK2T875N-expressing megakaryocytes (C,D). Cells were stained with propidium iodide (PI) and CD41⁺ cells were analyzed for ploidy content. Analyses were performed in duplicate and representative PI histograms are shown. Compared with JAK2WT (E), ploidy content in JAK2T875N megakaryocytes (F) was markedly reduced and an abnormal 6 n peak (arrow) was observed, consistent with a partial block at the initiation of the polyploidization process.