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Blood, Vol. 112, Issue 3, 875-885, August 1, 2008
Decreased differentiation of erythroid cells exacerbates ineffective erythropoiesis in β-thalassemia
Blood Libani et al.
112: 875
Supplemental materials for: Libani et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 5.37 MB)
- Figures S1. Characterization of the erythroid compartment shows an increase of homogeneous undifferentiated immature cell populations in thalassemic mice (JPG, 160 KB)
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FACS analysis of BM populations using CD71 and Ter119 co-staining. CD71+/Ter119+ and CD71−/Ter119+ staining identifies early (nucleated) and late (non-nucleated) erythroid cells1. The FACS profiles were further analyzed as described by Socolowsky2,3. In particular, the Ter119+ cells (indicated by the black oval) were analyzed by CD71/FSC, showing that the th3/+ mice exhibited a reduction of the most mature fractions (orthochromatic and nonŽnucleated indicated with red circles in wt mice) and an enrichment of the immature one (indicated with a green circle). Th3/th3 mice showed a complete absence of the mature fractions and a more uniform cell distribution as clearly indicated in the lower panel by FSC/SSC and morphological analyses (Figure 1A).
- Figures S2. Characterization of the erythroid compartment shows an increase of homogeneous undifferentiated immature cell populations in thalassemic mice (JPG, 140 KB)
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FACS analysis of splenic erythroid populations using CD71 and Ter119 co-staining. CD71+/Ter119+ and CD71−/Ter119+ staining identifies early (nucleated) and late (non-nucleated) erythroid cells1. The FACS profiles were further analyzed as described by Socolowsky2,3. In particular, the Ter119+ cells (indicated by the black oval) were analyzed by CD71/FSC, showing that the th3/+ mice exhibited a reduction of the most mature fractions (orthochromatic and nonŽnucleated indicated with red circles in wt mice) and an enrichment of the immature one (indicated with a green circle). The purified population indicates a representative FACS profile of splenocytes enriched in erythroid cell fractions by immunomagnetic lineage negative depletion (as described in the Material and Methods section). More than 95% purity was achieved in all cases.
- Figure S3. Characterization of erythroid cells from the livers of fetal thalassemic mice showed an increased number of immature erythroid cells similar to what is observed in adult thalassemic animals (JPG, 193 KB)
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The skewed FACS profile indicative of IE was already visible in th3/th3 embryos at E16.5: the mature erythroid population present in normal mice and reduced or absent in thalassemic mice is now indicated with an oval circle. This indicates that the BMT procedure faithfully transferred the genetic and phenotypic defect into normal adult mice. FACS analysis of (A) E14.5 and (B) E16.5 splenic erythroid populations using CD71 and Ter119 co-staining as described in Figure S1 and S2. Embryonic erythroid cells maintain CD71 expression even when enucleated (our observation).
- Figure S4. The majority of the Ki-67+ cells are also BrdU+ (JPG, 105 KB)
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In order to confirm that Ki-67+ cells were replicating, BrdU+ was injected in vivo and areas of EMH in liver sections from normal and thalassemic animals were investigated for BrdU and Ki-67 by immunofluorescence. Double positive cells were observed only rarely in +/+ liver sections, while many Ki-67+/BrdU+ cells were identified in specimens from thalassemic mice. Ki-67 (green)/BrdU (red)/Toto-3 (blue).
- Figures S5. TG101209 reduces splenomegaly both in 6 and 12 week old thalassemic mice (JPG, 35.2 KB)
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At the end of the treatment, Hb, spleen and erythropoiesis were investigated in th3/+ and wt animals treated with TG101209 or placebo. Hb levels, spleen weight, age of the animal and days of treatment are indicated.
- Figures S6. TG101209 reduces splenomegaly both in 6 and 12 week old thalassemic mice (JPG, 55.1 KB)
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At the end of the treatment, Hb, spleen and erythropoiesis were investigated in th3/+ and wt animals treated with TG101209 or placebo. The spleen of representative animals are shown. In figure S7 erythropoiesis was investigated using the CD71 and Ter119 antibodies in BM and spleen of 12 week old animals treated with TG101209 or placebo.
- Figures S7. TG101209 reduces splenomegaly both in 6 and 12 week old thalassemic mice (JPG, 133 KB)
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At the end of the treatment, Hb, spleen and erythropoiesis were investigated in th3/+ and wt animals treated with TG101209 or placebo. Erythropoiesis was investigated using the CD71 and Ter119 antibodies in BM and spleen of 12 week old animals treated with TG101209 or placebo.
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