Figure 2.
Mutant IDH1 impedes erythropoiesis in mice. (A) Left, flow cytometric determination of the proportions of CMP (CD16/32–CD34+), MEP (CD16/32–CD34–), and granulocyte-monocyte progenitors (GMP) (CD16/32+CD34+) among BM or spleen LK cells from control and IDH1-KI mice. Right, quantitation of the results in the left panel. Data are the mean ± SEM (n = 5). (B) Quantitation of numbers of colonies arising in cultures of nucleated BM or spleen cells that were isolated from control or IDH1-KI mice and cultured in BFU-E medium for 14 days or colony-forming unit-erythroid (CFU-E) medium for 2 days. Data are the mean ± SEM (n = 3). (C) Left, representative transmission electron micrographs of BM and spleen sections from control and IDH1-KI mice. Blue triangles, erythroblasts; red dots, reticulocytes and erythrocytes. Scale bars, 20 μm. Right, quantitation of the ratio of reticulocytes plus erythrocytes to erythroblasts in the left panels. Data are the mean ± SEM (n = 3). (D) Representative flow cytometric analysis of 4′,6-diamidino-2-phenylindole (DAPI) staining of Ter-119+ erythroid cells in BM and spleen of control and IDH1-KI mice (n = 3). (E) Left, flow cytometric analysis of erythroblasts in BM and spleen of control and IDH1-KI mice. Erythroblasts were divided into 4 subpopulations by surface staining for CD71 and Ter-119: Ter-119medCD71high proerythroblasts (S1), Ter-119highCD71high basophilic erythroblasts (S2), Ter-119highCD71med late basophilic and polychromatophilic erythroblasts (S3), and Ter-119highCD71low orthochromatophilic erythroblasts (S4). Right, quantitation of percentages of the S1 to S4 populations in the left panel. Data are the mean ± SEM (n = 5). (F) Representative images of erythroblasts at S1 to S4 stages (from panel E) stained with May-Grünwald-Giemsa (MGG; top) or 3,3′-diaminobenzidine (DAB)/hematoxylin (bottom) (n = 5). Scale bars, 10 μm. (G) Left, flow cytometric analysis of nucleated BM or spleen cells that were isolated from control and IDH1-KI mice, cultured in BFU-E medium for 14 days, and surface-stained to detect CD71 and Ter-119. Right, quantitation of proportions of the CD71–Ter-119–, CD71+Ter-119–, and CD71+Ter-119+ populations in the left panel. Data are the mean ± SEM of the collective results of 3 independent experiments. *P < .05; **P < .01; ***P < .001.

Mutant IDH1 impedes erythropoiesis in mice. (A) Left, flow cytometric determination of the proportions of CMP (CD16/32–CD34+), MEP (CD16/32–CD34–), and granulocyte-monocyte progenitors (GMP) (CD16/32+CD34+) among BM or spleen LK cells from control and IDH1-KI mice. Right, quantitation of the results in the left panel. Data are the mean ± SEM (n = 5). (B) Quantitation of numbers of colonies arising in cultures of nucleated BM or spleen cells that were isolated from control or IDH1-KI mice and cultured in BFU-E medium for 14 days or colony-forming unit-erythroid (CFU-E) medium for 2 days. Data are the mean ± SEM (n = 3). (C) Left, representative transmission electron micrographs of BM and spleen sections from control and IDH1-KI mice. Blue triangles, erythroblasts; red dots, reticulocytes and erythrocytes. Scale bars, 20 μm. Right, quantitation of the ratio of reticulocytes plus erythrocytes to erythroblasts in the left panels. Data are the mean ± SEM (n = 3). (D) Representative flow cytometric analysis of 4′,6-diamidino-2-phenylindole (DAPI) staining of Ter-119+ erythroid cells in BM and spleen of control and IDH1-KI mice (n = 3). (E) Left, flow cytometric analysis of erythroblasts in BM and spleen of control and IDH1-KI mice. Erythroblasts were divided into 4 subpopulations by surface staining for CD71 and Ter-119: Ter-119medCD71high proerythroblasts (S1), Ter-119highCD71high basophilic erythroblasts (S2), Ter-119highCD71med late basophilic and polychromatophilic erythroblasts (S3), and Ter-119highCD71low orthochromatophilic erythroblasts (S4). Right, quantitation of percentages of the S1 to S4 populations in the left panel. Data are the mean ± SEM (n = 5). (F) Representative images of erythroblasts at S1 to S4 stages (from panel E) stained with May-Grünwald-Giemsa (MGG; top) or 3,3′-diaminobenzidine (DAB)/hematoxylin (bottom) (n = 5). Scale bars, 10 μm. (G) Left, flow cytometric analysis of nucleated BM or spleen cells that were isolated from control and IDH1-KI mice, cultured in BFU-E medium for 14 days, and surface-stained to detect CD71 and Ter-119. Right, quantitation of proportions of the CD71–Ter-119–, CD71+Ter-119–, and CD71+Ter-119+ populations in the left panel. Data are the mean ± SEM of the collective results of 3 independent experiments. *P < .05; **P < .01; ***P < .001.

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