E beta-KD cells, a significantly lower percent of GPA(+)/CD71(-) cells was detected compared to control in culture day 18 cells (representative data shown in Figures 3C, 3D; triplicate experiments: GPA(+)/CD71(-); control = 28.165.8 vs. beta-KD = 1.660.5 , p = 0.02). On culture day 21, the cellular phenotypes were similar to those on culture day 18 suggesting the absence of further differentiationFigure 1. QPCR Quantitation of globin mRNA. RNA samples from erythroblasts cultured on day 14 were examined for globin mRNA expression using quantitative PCR. (A) Expression levels of beta-, gamma-, delta-, and epsilon-globins. (B) Expression levels of alpha-, mu, theta-, and zeta-globins. Average copy number per ng cDNA is shown on the y-axis from three separate donors, control (black bar) and betaKD (open bar). Standard deviation bars are shown in vertical lines. Asterisks signify statistical significance of p,0.05. doi:10.1371/journal.pone.0068307.gA Synthetic Model of Beta-ThalassemiaFigure 3. Flow cytometry analysis of terminal differentiation. Representative dot plots from (A) culture day 14 control erythroblasts, (B) culture day 14 beta-KD erythroblasts, (C) culture day 18 control erythroblasts, (D) culture day 18 beta-KD erythroblasts, (E) culture day 21 control erythroblasts, and (F) culture day 21 beta-KD erythroblasts. Cells were double stained with glycophorin A (GPA) and transferrin receptor (CD71). doi:10.1371/journal.pone.0068307.gWestern Analysis of Soluble and Membrane Insoluble Globin FractionsWestern analyses were performed to demonstrate the effects of beta-globin 23148522 chain imbalance upon alpha-, beta- and gammaglobin protein expression during terminal differentiation. Representative results are shown in Figure 4A of three separate donors. These results are consistent with 259869-55-1 reduced beta-globin gene expression, and beta-globin protein was also significantly reduced. Statisitcal analyses of Western blot band intensities 18055761 from three independent donors were compared for all globins and normalized to the loading control (beta-actin) on culture days 14, 18 and 21 (Table S2). The levels of cytosolic alpha-globin were significantly lower in the beta-KD cells; however, the level of reduction was less robust than that of beta-globin. Although gamma-globin was increased in the beta-KD samples, the increases did not reach statistical significance (Table S2). Since human alpha-globin chains do not assemble into soluble hemoglobin species, the globin chain imbalance caused by betathalassemia results in an excess of free alpha-globin chains. The excess alpha-globin chains lose their solubility and precipitate in the insoluble membrane fraction of erythrocytes and erythroblast precursor cells as a hallmark of the disease [15]. Those precipitates cause oxidative damage and contribute to the cellular PD 168393 site demise. To investigate whether the decreases in soluble alpha- globin chainsFigure 2. Hemoglobin and globin chain analyses. High performance liquid chromatography analyses of adult hemoglobin (HbA) and fetal hemoglobin (HbF) from culture day 21 erythroblasts (A) Control, (B) beta-KD. Total area under the (C) adult hemoglobin (HbA), and (D) fetal hemoglobin (HbF) peaks was measured using 1.56106 cultured cells from three donors. Each panel shows average values with standard deviation bars from control (black bar) and beta-KD (open bar). Cytospin preparations of the live cells were stained with Wright-Giemsa on culture day 21 for (E) control cells, (F).E beta-KD cells, a significantly lower percent of GPA(+)/CD71(-) cells was detected compared to control in culture day 18 cells (representative data shown in Figures 3C, 3D; triplicate experiments: GPA(+)/CD71(-); control = 28.165.8 vs. beta-KD = 1.660.5 , p = 0.02). On culture day 21, the cellular phenotypes were similar to those on culture day 18 suggesting the absence of further differentiationFigure 1. QPCR Quantitation of globin mRNA. RNA samples from erythroblasts cultured on day 14 were examined for globin mRNA expression using quantitative PCR. (A) Expression levels of beta-, gamma-, delta-, and epsilon-globins. (B) Expression levels of alpha-, mu, theta-, and zeta-globins. Average copy number per ng cDNA is shown on the y-axis from three separate donors, control (black bar) and betaKD (open bar). Standard deviation bars are shown in vertical lines. Asterisks signify statistical significance of p,0.05. doi:10.1371/journal.pone.0068307.gA Synthetic Model of Beta-ThalassemiaFigure 3. Flow cytometry analysis of terminal differentiation. Representative dot plots from (A) culture day 14 control erythroblasts, (B) culture day 14 beta-KD erythroblasts, (C) culture day 18 control erythroblasts, (D) culture day 18 beta-KD erythroblasts, (E) culture day 21 control erythroblasts, and (F) culture day 21 beta-KD erythroblasts. Cells were double stained with glycophorin A (GPA) and transferrin receptor (CD71). doi:10.1371/journal.pone.0068307.gWestern Analysis of Soluble and Membrane Insoluble Globin FractionsWestern analyses were performed to demonstrate the effects of beta-globin 23148522 chain imbalance upon alpha-, beta- and gammaglobin protein expression during terminal differentiation. Representative results are shown in Figure 4A of three separate donors. These results are consistent with reduced beta-globin gene expression, and beta-globin protein was also significantly reduced. Statisitcal analyses of Western blot band intensities 18055761 from three independent donors were compared for all globins and normalized to the loading control (beta-actin) on culture days 14, 18 and 21 (Table S2). The levels of cytosolic alpha-globin were significantly lower in the beta-KD cells; however, the level of reduction was less robust than that of beta-globin. Although gamma-globin was increased in the beta-KD samples, the increases did not reach statistical significance (Table S2). Since human alpha-globin chains do not assemble into soluble hemoglobin species, the globin chain imbalance caused by betathalassemia results in an excess of free alpha-globin chains. The excess alpha-globin chains lose their solubility and precipitate in the insoluble membrane fraction of erythrocytes and erythroblast precursor cells as a hallmark of the disease [15]. Those precipitates cause oxidative damage and contribute to the cellular demise. To investigate whether the decreases in soluble alpha- globin chainsFigure 2. Hemoglobin and globin chain analyses. High performance liquid chromatography analyses of adult hemoglobin (HbA) and fetal hemoglobin (HbF) from culture day 21 erythroblasts (A) Control, (B) beta-KD. Total area under the (C) adult hemoglobin (HbA), and (D) fetal hemoglobin (HbF) peaks was measured using 1.56106 cultured cells from three donors. Each panel shows average values with standard deviation bars from control (black bar) and beta-KD (open bar). Cytospin preparations of the live cells were stained with Wright-Giemsa on culture day 21 for (E) control cells, (F).
