American Society of Hematology

Figure 5

$Figure 5. Overexpression of functional miR-146 does not alter megakaryopoiesis. (A) Quantification of megakaryocytes in the bone marrow of transplanted mice. Bone marrow suspensions were stained with anti-CD42 and analyzed by flow cytometry. Similar results were obtained from the spleen (data not shown). Differences between GFP+ (P = .12) and GFP− groups (P = .23) were not significant. There were n = 5 mice analyzed from each group at 16 weeks after transplantation. (B) Megakaryocyte colony assays. Bone marrow cells were isolated 16 weeks after transplantation, fractionated by flow cytometry into c-kit+/GFP+ and c-kit+/GFP− subsets representing microRNA virus–infected and –uninfected populations, respectively, and analyzed in hematopoietic colony assays containing Tpo, Il-3, and IL-6. Colonies were enumerated at 7-8 days. Megakaryocytes were identified by staining with acetylthiocholiniodide. GM indicates granulocyte-macrophage; S Meg indicates small megakaryocytic colonies (3-5 cells); L Meg indicates large megakaryocytic colonies (> 5 cells); Mix indicates mixed colonies. There were n = 4 mice analyzed from each group. P > .1 for all groups. (C) Histology of hematopoietic tissues. Bone marrow and spleen from transplanted mice stained with hematoxylin-eosin. Tissue architecture, megakaryocyte numbers, and megakaryocyte morphology were similar in miR-146a–overexpressing and control mice. The mice shown here contained approximately 35% GFP-positive cells in bone marrow and spleen. Five similar pairs of mice were analyzed, and representative results are shown. Scale bars represent 20 μm. (D) Overexpressed miR-146a inhibits innate immune response in cultured macrophage. At 16 weeks transplantation, c-kit+/GFP+ bone marrow cells were purified by flow cytometry and cultured in media containing macrophage colony stimulating factor (M-CSF). After 7-8 days, the cells remained GFP+ and > 90% expressed the macrophage marker Mac1. The cells were stimulated with LPS for 16 hours and 1 day later harvested for RNA extraction. The expression of cytokine-encoding mRNAs Tnf (TNFα), Infb1 (interferon β), and Il1b (IL-1β) were analyzed by Q-RT-PCR. mRNA levels are plotted relative to levels in unstimulated cells, which were assigned an arbitrary value of 1. P values were .0001, .0008, and .0048, respectively. Results indicate triplicate RT-PCR assessments performed in 2 mice from each group.$

Overexpression of functional miR-146 does not alter megakaryopoiesis. (A) Quantification of megakaryocytes in the bone marrow of transplanted mice. Bone marrow suspensions were stained with anti-CD42 and analyzed by flow cytometry. Similar results were obtained from the spleen (data not shown). Differences between GFP⁺ (P = .12) and GFP⁻ groups (P = .23) were not significant. There were n = 5 mice analyzed from each group at 16 weeks after transplantation. (B) Megakaryocyte colony assays. Bone marrow cells were isolated 16 weeks after transplantation, fractionated by flow cytometry into c-kit⁺/GFP⁺ and c-kit⁺/GFP⁻ subsets representing microRNA virus–infected and –uninfected populations, respectively, and analyzed in hematopoietic colony assays containing Tpo, Il-3, and IL-6. Colonies were enumerated at 7-8 days. Megakaryocytes were identified by staining with acetylthiocholiniodide. GM indicates granulocyte-macrophage; S Meg indicates small megakaryocytic colonies (3-5 cells); L Meg indicates large megakaryocytic colonies (> 5 cells); Mix indicates mixed colonies. There were n = 4 mice analyzed from each group. P > .1 for all groups. (C) Histology of hematopoietic tissues. Bone marrow and spleen from transplanted mice stained with hematoxylin-eosin. Tissue architecture, megakaryocyte numbers, and megakaryocyte morphology were similar in miR-146a–overexpressing and control mice. The mice shown here contained approximately 35% GFP-positive cells in bone marrow and spleen. Five similar pairs of mice were analyzed, and representative results are shown. Scale bars represent 20 μm. (D) Overexpressed miR-146a inhibits innate immune response in cultured macrophage. At 16 weeks transplantation, c-kit+/GFP+ bone marrow cells were purified by flow cytometry and cultured in media containing macrophage colony stimulating factor (M-CSF). After 7-8 days, the cells remained GFP+ and > 90% expressed the macrophage marker Mac1. The cells were stimulated with LPS for 16 hours and 1 day later harvested for RNA extraction. The expression of cytokine-encoding mRNAs Tnf (TNFα), Infb1 (interferon β), and Il1b (IL-1β) were analyzed by Q-RT-PCR. mRNA levels are plotted relative to levels in unstimulated cells, which were assigned an arbitrary value of 1. P values were .0001, .0008, and .0048, respectively. Results indicate triplicate RT-PCR assessments performed in 2 mice from each group.

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