Effects of Ezh2 deletion on hematopoietic precursors and progenitors in mice expressing Jak2V617F. (A) Representative dot plots of flow cytometric analysis of erythroid precursors in the BM and spleens of control, Ezh2^−/−, Jak2^VF/+, and Jak2^VF/+ Ezh2^−/− mice (at 24 weeks after pI-pC injection) using surface markers CD71 and Ter119. Percentages of erythroid precursor cells at different stages of differentiation (stages I-IV, from immature to mature) are shown in bar graphs as mean ± SEM (n = 5-8). (B) Representative dot plots of flow cytometric analysis of megakaryocytic precursors in the BM and spleens of control, Ezh2^−/−, Jak2^VF/+, and Jak2^VF/+ Ezh2^−/− mice using CD41 and CD61 staining. CD41⁺CD61⁺ megakaryocytic precursors in the BM and spleens are shown in bar graphs as mean ± SEM (n = 5-8). (C) Percentages of LSKs (Lin⁻Sca-1⁺c-kit⁺), LT-HSCs (Lin⁻Sca-1⁺c-kit⁺CD34⁻CD135⁻), ST-HSCs (Lin⁻Sca-1⁺c-kit⁺CD34⁺CD135⁻), MPPs (Lin⁻Sca-1⁺c-kit⁺CD34⁺CD135⁺), CMPs (Lin⁻Sca-1⁻c-kit⁺CD34⁺FcγRII/II^low), GMPs (Lin⁻Sca-1⁻c-kit⁺CD34⁺FcγRII/II^high), and MEPs (Lin⁻Sca-1⁻c-kit⁺CD34⁻FcγRII/III⁻) in the BM and spleens from control, Ezh2^−/−, Jak2^VF/+, and Jak2^VF/+ Ezh2^−/− at 24 weeks after pI-pC are shown in bar graphs as mean ± SEM (n = 5-12). (D) Total numbers of LSKs, LT-HSCs, ST-HSCs, MPPs, CMPs, GMPs, and MEPs in the BM and spleens are shown in bar graphs as mean ± SEM (n = 5-12). (E) BM (2 × 10⁴) or spleen cells (1 × 10⁵) from control, Ezh2^−/−, Jak2^VF/+, and Jak2^VF/+ Ezh2^−/− mice (n = 4-7) were plated in methylcellulose medium supplemented with cytokines. Burst-forming unit–erythroid (BFU-E), CFU-granulocyte/macrophage (CFU-GM), and CFU–granulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM) colonies were scored 7 days after plating. (F) CFU-erythroid (CFU-E) colonies. BM or spleen cells (1 × 10⁵) from control, Ezh2^−/−, Jak2^VF/+, and Jak2^VF/+ Ezh2^−/− mice (n = 5-7) were plated in methylcellulose medium in the absence of erythropoietin. CFU-E colonies were scored after 2 days. (G) CFU-Mk colonies. BM cells (1 × 10⁵) from control, Ezh2^−/−, Jak2^VF/+, and Jak2^VF/+ Ezh2^−/− mice (n = 5-7) were plated into collagen-based MegaCult medium supplemented with interleukin (IL)-3, IL-6, IL-11, and thrombopoietin. Megakaryocytic (CFU-Mk) colonies were assessed after culturing for 8 days. All data are shown as mean ± SEM. One-way ANOVA was used for comparisons of all 4 groups of mice, and the Student t test was used to compare between 2 groups of mice (*P < .05; **P < .005; ns, not significant).