American Society of Hematology

Figure 2.

$miR-144/451 disruption reduces the accumulation of insoluble α-globin and reactive oxygen species (ROS) in β-thalassemia. (A) Soluble and insoluble RBC globin proteins. Equal volumes of RBCs (normalized by hematocrit) were lysed, centrifuged to separate insoluble and soluble proteins, fractionated by Triton–acetic acid–urea (TAU) gel electrophoresis to resolve α- and β-globin proteins, and stained with Coomassie brilliant blue. (B) Results of multiple experiments performed as described in panel A. The y-axis represents the relative levels of insoluble α-globin, as measured via automated image analysis of TAU gels, and expressed in arbitrary units. n = 3 to 6 mice for each genotype. (C) Thiazole orange–stained reticulocytes were isolated by fluorescence-activated cell sorting to a purity of ∼95%, then analyzed for electron-dense α-globin precipitates by transmission electron microscopy using an FEI Tecnai 200Kv FEG Transmission Electron Microscope with an ATM XR41 digital camera. Representative micrographs show reticulocytes from mice with the indicated genotypes. Bars represent 2 μm. (D) Areas of α-globin inclusions in reticulocytes (y-axis) determined by automated image analysis of electron micrographs prepared in the same manner as those in panel C. n = 4 to 8 mice for each genotype. Approximately 200 cells from each mouse were analyzed. (E) Flow cytometry quantification of ROS, determined by 2′, 7′-dichlorodihydrofluorescein diacetate staining of bone marrow erythroblasts. Ery.A, Ery.B, and Ery.C represent distinct developmental stages defined by the expression of erythroid maturation markers and cell size (supplemental Figure 2). n = 6 to 10 mice for each genotype. Bar charts show data as mean values ± SD; ∗∗∗∗P < .0001; ∗∗P < .01; ∗P < .05; ns, not significant.$

miR-144/451 disruption reduces the accumulation of insoluble α-globin and reactive oxygen species (ROS) in β-thalassemia. (A) Soluble and insoluble RBC globin proteins. Equal volumes of RBCs (normalized by hematocrit) were lysed, centrifuged to separate insoluble and soluble proteins, fractionated by Triton–acetic acid–urea (TAU) gel electrophoresis to resolve α- and β-globin proteins, and stained with Coomassie brilliant blue. (B) Results of multiple experiments performed as described in panel A. The y-axis represents the relative levels of insoluble α-globin, as measured via automated image analysis of TAU gels, and expressed in arbitrary units. n = 3 to 6 mice for each genotype. (C) Thiazole orange–stained reticulocytes were isolated by fluorescence-activated cell sorting to a purity of ∼95%, then analyzed for electron-dense α-globin precipitates by transmission electron microscopy using an FEI Tecnai 200Kv FEG Transmission Electron Microscope with an ATM XR41 digital camera. Representative micrographs show reticulocytes from mice with the indicated genotypes. Bars represent 2 μm. (D) Areas of α-globin inclusions in reticulocytes (y-axis) determined by automated image analysis of electron micrographs prepared in the same manner as those in panel C. n = 4 to 8 mice for each genotype. Approximately 200 cells from each mouse were analyzed. (E) Flow cytometry quantification of ROS, determined by 2′, 7′-dichlorodihydrofluorescein diacetate staining of bone marrow erythroblasts. Ery.A, Ery.B, and Ery.C represent distinct developmental stages defined by the expression of erythroid maturation markers and cell size (supplemental Figure 2). n = 6 to 10 mice for each genotype. Bar charts show data as mean values ± SD; ∗∗∗∗P < .0001; ∗∗P < .01; ∗P < .05; ns, not significant.

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