Figure 3.
IDH1 mutation causes heme deficiency in erythroid cells. (A) Immunoblot to detect the indicated hemoglobin proteins in Ter-119+ and Ter-119– nucleated cells from BM and spleen of control and IDH1-KI mice (n = 3). Ponceau S, loading control. (B) Left, low (top) and high (insets; bottom) magnification views of 3,3′-diaminobenzidine/hematoxylin-stained sections of BM and spleen from control and IDH1-KI mice. Scale bars, 50 μm. Right, quantitation of 3,3′-diaminobenzidine staining intensities in the left panels. Data are the mean ± SEM (n = 4). (C-D) Quantitation of heme content as determined by fluorescence spectroscopy in lineage+, lineage–, Ter-119+, and Ter-119– nucleated BM cells (C) and in Ter-119+ and Ter-119– nucleated spleen cells (D) from control and IDH1-KI mice. Data are the mean ± SEM (n = 4). (E) Top and middle, representatitve May-Grünwald-Giemsa staining of K562 cells expressing WT or mutant IDH1 that were left uninduced (top) or induced with hydroxyurea plus sodium butyrate (middle). Bottom, cell pellets of the cells in the middle panel. Scale bars, 50 μm. (F) Representative flow cytometric analysis of CD235a expression by the induced K562 cells in panel E (n = 5). (G) Quantitation of heme content in the induced cells in panel E determined as in panel C (n = 5). (H) Immunoblot to detect the indicated hemoglobin proteins in the induced K562 cells in panel E (n = 3). β-actin, loading control. (I) Microscopic view of nontransfected K562 cells that were treated with either hemin or SA (heme synthesis inhibitor) and stained by using the WG-KI biochemistry method to detect peroxidase particles (n = 5). Scale bars, 10 μm. (J) Relative mRNA levels of GATA1, CEBPA, SPI1, and GATA2 in the K562 cells in panel I. Data are the mean ± SEM (n = 4). β-actin, endogenous control. (K) Relative mRNA levels of GATA1, CEBPA, SPI1, and GATA2 in BM CMPs from control and IDH1-KI mice. Data are the mean ± SEM (n = 4). β-actin, endogenous control. (L) Relative mRNA levels of GATA1, CEBPA, SPI1, and GATA2 in K562 cells overexpressing WT or mutant IDH1.Data are the mean ± SEM (n = 4). *P < .05; **P < .01; ***P < .001. β-actin, endogenous control. TF, transcription factor.

IDH1 mutation causes heme deficiency in erythroid cells. (A) Immunoblot to detect the indicated hemoglobin proteins in Ter-119+ and Ter-119– nucleated cells from BM and spleen of control and IDH1-KI mice (n = 3). Ponceau S, loading control. (B) Left, low (top) and high (insets; bottom) magnification views of 3,3′-diaminobenzidine/hematoxylin-stained sections of BM and spleen from control and IDH1-KI mice. Scale bars, 50 μm. Right, quantitation of 3,3′-diaminobenzidine staining intensities in the left panels. Data are the mean ± SEM (n = 4). (C-D) Quantitation of heme content as determined by fluorescence spectroscopy in lineage+, lineage–, Ter-119+, and Ter-119– nucleated BM cells (C) and in Ter-119+ and Ter-119– nucleated spleen cells (D) from control and IDH1-KI mice. Data are the mean ± SEM (n = 4). (E) Top and middle, representatitve May-Grünwald-Giemsa staining of K562 cells expressing WT or mutant IDH1 that were left uninduced (top) or induced with hydroxyurea plus sodium butyrate (middle). Bottom, cell pellets of the cells in the middle panel. Scale bars, 50 μm. (F) Representative flow cytometric analysis of CD235a expression by the induced K562 cells in panel E (n = 5). (G) Quantitation of heme content in the induced cells in panel E determined as in panel C (n = 5). (H) Immunoblot to detect the indicated hemoglobin proteins in the induced K562 cells in panel E (n = 3). β-actin, loading control. (I) Microscopic view of nontransfected K562 cells that were treated with either hemin or SA (heme synthesis inhibitor) and stained by using the WG-KI biochemistry method to detect peroxidase particles (n = 5). Scale bars, 10 μm. (J) Relative mRNA levels of GATA1, CEBPA, SPI1, and GATA2 in the K562 cells in panel I. Data are the mean ± SEM (n = 4). β-actin, endogenous control. (K) Relative mRNA levels of GATA1, CEBPA, SPI1, and GATA2 in BM CMPs from control and IDH1-KI mice. Data are the mean ± SEM (n = 4). β-actin, endogenous control. (L) Relative mRNA levels of GATA1, CEBPA, SPI1, and GATA2 in K562 cells overexpressing WT or mutant IDH1.Data are the mean ± SEM (n = 4). *P < .05; **P < .01; ***P < .001. β-actin, endogenous control. TF, transcription factor.

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