Figure 1
Figure 1. In β-thalassemic mice, there is a decreased number of differentiated cells, a small increase of apoptotic and hemolytic markers, and elevated production of Epo. (A) In cytospins of purified splenic erythroid cells, distinctive types of cells can be seen representing different stages of maturation. More mature cells are characterized by a smaller size, decreased cytoplasmic basophilia, and an increase in nuclear pyknosis. (May-Grünwald Giemsa stain; magnification, 400×.) These observations were corroborated by FACS analyses (Figures S1,S2). Apoptosis was investigated (B) by CC3 assay on spleen sections; (C) by TUNEL assay on BM and spleen sections; (D) by CC3 assay on purified erythroid cells; and (E) by annexin-V assay on fresh spleen and BM cells (data not shown; n ≥ 3 per genotype). For panels A through D, images were captured on a Nikon Eclipse E800 microscope (Melville, NY), with a Retiga Exi camera (Qimaging, Burnaby, BC) and a Plan Fluor 40×/0.75 numeric aperture objective, then acquired using the IPLab 3.65a software (Scanalytics, Fairfax, VA). Brightness/contrast and color balance were adjusted using Adobe Photoshop 7.0.1 (Adobe Systems, San Jose, CA). The levels of hemolytic markers (F) bilirubin and (G) LDH were also investigated (n ≥ 6 per genotype). In panels E-G, th3/+ and th3/th3 mice are indicated as +/− and −/−, respectively. A nonparametric t test was used for statistical analysis. Increased Epo levels inversely correlate with those of hemoglobin in thalassemic mice. Measurements were made of (H) Epo levels in mice 2 months after BMT and (I) Epo and Hb levels in mice up to 1 year of age. In panel H, a nonparametric t test was used for statistical analysis; n ≥ 3 per genotype; P = .037 (*) and P = .001 (***), respectively, for th3/+ and th3/th3 mice compared with wt animals. In this panel th3/+ and th3/th3 mice are indicated, respectively, as +/− and −/−. In panel I, increased Epo levels inversely correlate with Hb in thalassemic mice. Epo levels were measured in random mice up to 1 year of age or 1 year after BMT in wt (□, n = 17) and th3/+ (▲, n = 18) mice. Pearson r test was used to determine the degree of linear association or the correlation coefficient between the Hb and Epo levels (wt, nonsignificant, P = .087; th3/+, P = .027). Error bars represent SD.

In β-thalassemic mice, there is a decreased number of differentiated cells, a small increase of apoptotic and hemolytic markers, and elevated production of Epo. (A) In cytospins of purified splenic erythroid cells, distinctive types of cells can be seen representing different stages of maturation. More mature cells are characterized by a smaller size, decreased cytoplasmic basophilia, and an increase in nuclear pyknosis. (May-Grünwald Giemsa stain; magnification, 400×.) These observations were corroborated by FACS analyses (Figures S1,S2). Apoptosis was investigated (B) by CC3 assay on spleen sections; (C) by TUNEL assay on BM and spleen sections; (D) by CC3 assay on purified erythroid cells; and (E) by annexin-V assay on fresh spleen and BM cells (data not shown; n ≥ 3 per genotype). For panels A through D, images were captured on a Nikon Eclipse E800 microscope (Melville, NY), with a Retiga Exi camera (Qimaging, Burnaby, BC) and a Plan Fluor 40×/0.75 numeric aperture objective, then acquired using the IPLab 3.65a software (Scanalytics, Fairfax, VA). Brightness/contrast and color balance were adjusted using Adobe Photoshop 7.0.1 (Adobe Systems, San Jose, CA). The levels of hemolytic markers (F) bilirubin and (G) LDH were also investigated (n ≥ 6 per genotype). In panels E-G, th3/+ and th3/th3 mice are indicated as +/− and −/−, respectively. A nonparametric t test was used for statistical analysis. Increased Epo levels inversely correlate with those of hemoglobin in thalassemic mice. Measurements were made of (H) Epo levels in mice 2 months after BMT and (I) Epo and Hb levels in mice up to 1 year of age. In panel H, a nonparametric t test was used for statistical analysis; n ≥ 3 per genotype; P = .037 (*) and P = .001 (***), respectively, for th3/+ and th3/th3 mice compared with wt animals. In this panel th3/+ and th3/th3 mice are indicated, respectively, as +/− and −/−. In panel I, increased Epo levels inversely correlate with Hb in thalassemic mice. Epo levels were measured in random mice up to 1 year of age or 1 year after BMT in wt (□, n = 17) and th3/+ (▲, n = 18) mice. Pearson r test was used to determine the degree of linear association or the correlation coefficient between the Hb and Epo levels (wt, nonsignificant, P = .087; th3/+, P = .027). Error bars represent SD.

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