Loss of Mcl-1 does not affect platelet production. (A) Western blot of protein lysates from platelets and fetal liver–derived megakaryocytes. E13.5 fetal liver cells were cultured in SFM in thrombopoietin for 5 days, and then large megakaryocytes were purified from a BSA gradient. Mcl-1 was efficiently deleted in mouse megakaryocytes. Its loss did not influence Bcl-x_L protein levels. Mcl-1 expression was below detection level in control blood platelets under the conditions used. Δ/Δ indicates platelet factor 4 (Pf4) Cre-specific deletion. Actin was used as a control for protein loading. Negative control for Bcl-x_L was Bcl-x^−/− mouse embryonic fibroblast (MEF) lysate. (B) Blood platelet counts were normal in Mcl-1^Pf4Δ/Pf4Δ mice and Mcl-1^fl/fl control mice at 7 weeks of age. Data represent mean ± SD. Each symbol represents an individual mouse. (C) Platelet survival curves in Mcl-1^Pf4Δ/Pf4Δ, Mcl-1^fl/fl, and Mcl-1^+/+ mice. Platelets were labeled via IV injection of a DyLight 488–conjugated anti-GPIbβ (CD42c) Ab. Data represent mean ± SD n = 4 Mcl-1^+/+; n = 5 Mcl-1^Pf4Δ/Pf4Δ; n = 1 Mcl-1^fl/fl mice per group. (D) Morphologically recognizable megakaryocytes in H&E-stained sections of BM from Mcl-1^Pf4Δ/Pf4Δ and Mcl-1^fl/fl mice. Each symbol represents the mean number per field of view (FOV; ×200) from 5 fields per individual mouse. Data represent overall mean ± SD. (E) Ploidy distribution profile of CD41⁺ BM cells in Mcl-1^Pf4Δ/Pf4Δ and Mcl-1^fl/fl mice determined by flow cytometry. Data represent mean ± SD; n = 4-6 mice per genotype. (F) Serum thrombopoietin levels in Mcl-1^Pf4Δ/Pf4Δ and Mcl-1^fl/fl mice. Each symbol represents an individual mouse. Data represent mean ± SD. (G) Hematopoietic progenitor cell numbers in adult mice. 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 = 2 Mcl-1^fl/fl; n = 3, Mcl-1^Pf4Δ/Pf4Δ. (H) Proplatelet formation in cultured megakaryocytes (MK). E13.5 fetal liver cells were cultured in SFM in thrombopoietin for 3 days. On day 4, large megakaryocytes were purified from a BSA gradient and reseeded to investigate proplatelet formation. Data are presented as the percentage of proplatelet forming megakaryocytes per day and represent mean ± SEM, n = 5 Mcl-1^+/+; n = 5, Mcl-1^Pf4Δ/Pf4Δ; n = 3, Mcl-1^fl/fl biological replicates. (I) Blood platelet counts in response to antiplatelet serum (APS)–induced thrombocytopenia. No significant difference was detected. Data represent mean ± SD; n = 5 mice per genotype at each time point.