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Blood, Vol. 112, Issue 9, 3878-3888, November 1, 2008
Stat5 regulates cellular iron uptake of erythroid cells via IRP-2 and TfR-1
Blood Kerenyi et al.
112: 3878
Supplemental materials for: Kerenyi et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 95.8 KB)
- Table S1. Real time PCR and ChIP primers (PDF, 264 KB)
- Figure S1. Erythrocytes from Stat5−/− newborn mice are microcytic and hypochromic (JPG, 63.9 KB)
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(A) Blood smears from newborn wt and Stat5−/− mice stained with benzidine (stains hemoglobin). (B) Blood smears from newborn wt and Stat5−/− mice stained with Wright-Giemsa. (C) Frequency of BFU-E colonies from wt versus Stat5−/− fetal liver cells (data are presented as mean ± SD; n=4).
- Figure S2. Stat5−/− erythroid cells proliferate like wt cells (JPG, 65.2 KB)
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(A) Pregnant Stat5+/− females were pulsed with BrdU for 1h, and fetal livers were isolated. Typical flow-cytometry data for Ter119/BrdU double positive fetal livers from wt embryos (left) and corresponding quantification of wt versus Stat5-deficient cells (right; data presented as mean ± SD; n=4) is shown. (B) Fetal liver erythroblasts grown under self-renewal conditions from wt and Stat5−/− embryos were stained with CFSE. Dilution of fluorescence over time was analyzed by flow cytometry as indicated as a measure of proliferation rates. Representative histograms taken from three independently performed experiments are depicted. (C) Western blot analysis for PCNA with lysates from primary erythroblast derived from wt and Stat5−/− fetal livers under self-renewal is depicted. Actin was used as loading control.
- Figure S3. Stat5−/− embryos show an increase of MEPs in the fetal liver (JPG, 60.8 KB)
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E13.5 fetal livers were isolated and stained according to6,7 for CMP (common myeloid progenitor) GMP (granulocytic/monocytic progenitor) and MEP (megakaryocytic /erythroid progenitor). Note the increase of the MEP population in Stat5−/− fetal livers (data are presented as mean ± SD; n=3–5).
- Figure S4. No apparent morphological differences between wt and Stat5−/− erythroid progenitors (JPG, 61.4 KB)
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(A) Wt and Stat5−/− erythroid progenitors were sorted from fetal livers and sub-fractioned according to their cell surface marker phenotype (R2: TfR-1+/ Ter119lo; R3: TfR-1+/Ter119hi; R4+R5: TfR-1lo/− Ter119hi). Aliquots of the respective sub-fractions were cyto-centrifuged onto glass slides and subsequently stained with either May-Grunwald Giemsa (left panels) or Benzidine-Wright Giemsa (right panels).
- Figure S5. Increased expression of ferritin in Stat5−/− primary erythroblasts (JPG, 20.7 KB)
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Lysates from wt and Stat5−/− erythroblasts were subjected to Western blot analysis for ferritin. Total-eIF2-alpha was used as loading control.
- Figure S6. Increased eIF2-alpha phosphorylation in Stat5−/− primary erythroblasts (JPG, 31 KB)
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Lysates from wt and Stat5−/− erythroblasts were subjected to Western blot analysis. Total-eIF4E was used as loading control.
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