Figure 2
Figure 2. ABT-737 induces cell death in Mcl-1–deficient megakaryocytes. (A) Caspase 3/7 activity in fetal liver–derived megakaryocytes treated with ABT-737. Fetal livers were cultured in SFM plus TPO for 3 days. Large megakaryocytes were purified from a BSA gradient and reseeded. Caspase activity was measured 5 hours after treatment with ABT-737 (5μM) or vehicle (DMSO) with or without the caspase inhibitor, QVD, using the Promega Caspase-Glo assay. Data represent fold increase compared with control ± SEM, n = 2 technical replicates for each genotype. Representative of 4-5 independent experiments. (B) Mitochondrial function in fetal liver–derived megakaryocytes treated with ABT-737. ATP levels in fetal liver–derived megakaryocytes were measured 24 hours after reseeding and treating with ABT-737 (5μM) or vehicle (DMSO) using the CellTiter-Glo assay system. DMSO control was set as 100% for each genotype. Data represents mean ± SEM, n = 4-9 independent experiments. ***P < .0001. (C) Peripheral blood platelet counts in Mcl-1+/+, Mcl-1fl/fl, and Mcl-1Pf4Δ/Pf4Δ mice treated with a single IP injection of ABT-737 (75 mg/kg). Cohorts of mice were bled at indicated time points to determine platelet counts; n = 3-6 mice per genotype at 2 hours after treatment, n = 3-8 at 6 hours, n = 5-9 at 24 hours and n = 6-7 at 48 hours. Data represent mean ± SD. (D) Megakaryocyte counts in Mcl-1+/+, Mcl-1fl/fl, and Mcl-1Pf4Δ/Pf4Δ mice treated with a single IP injection of ABT-737 (75 mg/kg). Data represent mean number of BM megakaryocytes per field of view (FOV; ×200) from at least 5 fields per individual mouse; n = 3-6 mice per genotype at each time point. Data represent mean ± SD. (E) Hematopoietic progenitor cell numbers in adult mice 2.5 hours after treatment with ABT-737. Twenty-five thousand BM cells were cultured with stem cell factor, IL-3, and erythropoietin in semisolid agar for 7 days. Non-megakaryocyte (MK) colonies represent the total of blast, granulocyte, mixed granulocyte/macrophage, macrophage, and eosinophil colonies. Data represent ± SEM; n = 3, Mcl-1+/+; n = 3, Mcl-1Pf4Δ/Pf4Δ.

ABT-737 induces cell death in Mcl-1–deficient megakaryocytes. (A) Caspase 3/7 activity in fetal liver–derived megakaryocytes treated with ABT-737. Fetal livers were cultured in SFM plus TPO for 3 days. Large megakaryocytes were purified from a BSA gradient and reseeded. Caspase activity was measured 5 hours after treatment with ABT-737 (5μM) or vehicle (DMSO) with or without the caspase inhibitor, QVD, using the Promega Caspase-Glo assay. Data represent fold increase compared with control ± SEM, n = 2 technical replicates for each genotype. Representative of 4-5 independent experiments. (B) Mitochondrial function in fetal liver–derived megakaryocytes treated with ABT-737. ATP levels in fetal liver–derived megakaryocytes were measured 24 hours after reseeding and treating with ABT-737 (5μM) or vehicle (DMSO) using the CellTiter-Glo assay system. DMSO control was set as 100% for each genotype. Data represents mean ± SEM, n = 4-9 independent experiments. ***P < .0001. (C) Peripheral blood platelet counts in Mcl-1+/+, Mcl-1fl/fl, and Mcl-1Pf4Δ/Pf4Δ mice treated with a single IP injection of ABT-737 (75 mg/kg). Cohorts of mice were bled at indicated time points to determine platelet counts; n = 3-6 mice per genotype at 2 hours after treatment, n = 3-8 at 6 hours, n = 5-9 at 24 hours and n = 6-7 at 48 hours. Data represent mean ± SD. (D) Megakaryocyte counts in Mcl-1+/+, Mcl-1fl/fl, and Mcl-1Pf4Δ/Pf4Δ mice treated with a single IP injection of ABT-737 (75 mg/kg). Data represent mean number of BM megakaryocytes per field of view (FOV; ×200) from at least 5 fields per individual mouse; n = 3-6 mice per genotype at each time point. Data represent mean ± SD. (E) Hematopoietic progenitor cell numbers in adult mice 2.5 hours after treatment with ABT-737. Twenty-five thousand BM cells were cultured with stem cell factor, IL-3, and erythropoietin in semisolid agar for 7 days. Non-megakaryocyte (MK) colonies represent the total of blast, granulocyte, mixed granulocyte/macrophage, macrophage, and eosinophil colonies. Data represent ± SEM; n = 3, Mcl-1+/+; n = 3, Mcl-1Pf4Δ/Pf4Δ.

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