Figure 1.
Effective erythropoiesis in th3+/− mice upon the depletion of miR-144/451. (A) Automated hematology analysis showing the elevated RBC, Hb, and HCT levels, and the reduced RDW in mKO/th3+/− mice. Reticulocytes were detected using flow cytometer. (B) Statistical analysis of the spleen weight. n = 8 to 10. Representative images of spleens from each group are shown. (C) HE staining and IHC staining of cell surface marker Ter119 in spleen sections of 10-week-old mice. Relatively normal splenic architecture in mKO/th3+/− mice is shown. (D) Flow cytometric analysis of the frequencies of splenic erythroblasts, reticulocytes, and mature RBCs identified using Ter119, CD44, and forward scatter channel (FSC). Effective erythropoiesis in mKO/th3+/− mice is evidenced by the significantly declined numbers of erythroblasts (Q1, Ter119+/CD44+/FSCHi) and increased proportion of mature erythrocytes (Q3, Ter119+/CD44-/FSCLow). (E) Observation of Heinz bodies, erythrocyte morphology, and the frequency of reticulocytes in peripheral blood from different groups of mice. Heinz bodies, reflecting precipitated α-globin on erythrocyte membranes, are reduced in mKO/th3+/− erythrocytes. Wright-Giemsa stain shows the significantly decreased anisocytosis and polychromasia (blue tinge) in peripheral blood from mKO/th3+/− mice compared with th3+/− mice. mKO/th3+/− mice exhibit less reticulocytes in circulating blood compared with th3+/− mice. (F) Flow cytometry analysis of the percentages of reticulocytes in peripheral blood from 4 genotypic mice. The frequency of both early-stage (R1, Ter119HiCD71Hi) and late-stage reticulocytes (R2, Ter119HiCD71Med) are declined, and the percentage of mature RBCs (R3, Ter119HiCD71Neg) is increased in the circulating blood from mKO/th3+/− mice compared with that in the blood from th3+/− mice. (A-F) All experiments were repeated at least 3 times. (G) Erythrocytes in mKO/th3+/− mice survive longer than erythrocytes in th3+/− mice. In vivo biotin labeling was used to follow erythrocyte survival kinetics over a 40-day period. The x-axis shows the days after biotin labeling; the y-axis shows the percentages of the biotinylated RBCs in mice at the age of 3 months. The time required for loss of 50% of the labeled RBCs in mKO/th3+/− mice is 6 days longer than in th3+/− mice. Experiments were repeated twice. (H) PGSK stain showing iron levels in reticulocytes of peripheral blood. mKO/th3+/− reticulocytes contain less iron. Experiments were repeated at least 3 times. (I) Aged mKO/th3+/− mice still possess low spleen weight to body weight ratio, suggesting a persistent alleviation of thalassemia because of the loss of miR-144/451. ∗P < .05; ∗∗P < .01. Hb, hemoglobin; HCT, hematocrit; HE, Hematoxylin/eosin; IHC, immunohistochemical staining; PGSK, phen green SK diacetate; RDW, red cell distribution width.

Effective erythropoiesis in th3+/− mice upon the depletion of miR-144/451. (A) Automated hematology analysis showing the elevated RBC, Hb, and HCT levels, and the reduced RDW in mKO/th3+/− mice. Reticulocytes were detected using flow cytometer. (B) Statistical analysis of the spleen weight. n = 8 to 10. Representative images of spleens from each group are shown. (C) HE staining and IHC staining of cell surface marker Ter119 in spleen sections of 10-week-old mice. Relatively normal splenic architecture in mKO/th3+/− mice is shown. (D) Flow cytometric analysis of the frequencies of splenic erythroblasts, reticulocytes, and mature RBCs identified using Ter119, CD44, and forward scatter channel (FSC). Effective erythropoiesis in mKO/th3+/− mice is evidenced by the significantly declined numbers of erythroblasts (Q1, Ter119+/CD44+/FSCHi) and increased proportion of mature erythrocytes (Q3, Ter119+/CD44-/FSCLow). (E) Observation of Heinz bodies, erythrocyte morphology, and the frequency of reticulocytes in peripheral blood from different groups of mice. Heinz bodies, reflecting precipitated α-globin on erythrocyte membranes, are reduced in mKO/th3+/− erythrocytes. Wright-Giemsa stain shows the significantly decreased anisocytosis and polychromasia (blue tinge) in peripheral blood from mKO/th3+/− mice compared with th3+/− mice. mKO/th3+/− mice exhibit less reticulocytes in circulating blood compared with th3+/− mice. (F) Flow cytometry analysis of the percentages of reticulocytes in peripheral blood from 4 genotypic mice. The frequency of both early-stage (R1, Ter119HiCD71Hi) and late-stage reticulocytes (R2, Ter119HiCD71Med) are declined, and the percentage of mature RBCs (R3, Ter119HiCD71Neg) is increased in the circulating blood from mKO/th3+/− mice compared with that in the blood from th3+/− mice. (A-F) All experiments were repeated at least 3 times. (G) Erythrocytes in mKO/th3+/− mice survive longer than erythrocytes in th3+/− mice. In vivo biotin labeling was used to follow erythrocyte survival kinetics over a 40-day period. The x-axis shows the days after biotin labeling; the y-axis shows the percentages of the biotinylated RBCs in mice at the age of 3 months. The time required for loss of 50% of the labeled RBCs in mKO/th3+/− mice is 6 days longer than in th3+/− mice. Experiments were repeated twice. (H) PGSK stain showing iron levels in reticulocytes of peripheral blood. mKO/th3+/− reticulocytes contain less iron. Experiments were repeated at least 3 times. (I) Aged mKO/th3+/− mice still possess low spleen weight to body weight ratio, suggesting a persistent alleviation of thalassemia because of the loss of miR-144/451. ∗P < .05; ∗∗P < .01. Hb, hemoglobin; HCT, hematocrit; HE, Hematoxylin/eosin; IHC, immunohistochemical staining; PGSK, phen green SK diacetate; RDW, red cell distribution width.

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