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Blood, Vol. 111, Issue 9, 4511-4522, May 1, 2008
Stat5 activation enables erythropoiesis in the absence of EpoR and Jak2
Blood Grebien et al.
111: 4511
Supplementary materials for: Grebien et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 78.4 KB)
- Table S1. Cultured Jak2−/−- and EpoR−/−-cS5 cells represent homogenous populations of pro-erythroblasts (JPG, 28.9 KB)
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Cultured fetal liver cells (WT-GFP and WT-cS5 as well as EpoR−/−-cS5 and Jak2−/−-cS5 cells) were expanded for 9 days and subjected to FACS analysis for different lineage markers. All values (% marker positive, mean +/− SD of 3 independent determinations) are derived from GFP+ gated (i.e. cS5-expressing) cells.
- Figure S1. cS5-expressing Jak2−/− and EpoR−/− cells form normal CFU-E colonies (JPG, 49.9 KB)
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WT, Jak2−/− and EpoR−/− fetal liver cells were transduced with GFP or cS5 and subjected to CFU-E assays and representative colonies photographed after 2 days.
- Figure S2. Expression of WT-Stat5 or Bcl-xL is unable to rescue the erythroid defect of Jak2−/− and EpoR−/− cells (JPG, 62.6 KB)
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(A) WT, Jak2−/− and EpoR−/− fetal liver cells expressing GFP, Stat5 or Bcl-xL were subjected to CFU-E assays or BFU-E assays (B). Benzidine-positive colonies were scored at day 2 (CFU-E) or 8 (BFU-E) (n=3).
- Figure S3. Molecular characterization of Stat5a-ER* and cS5-ER* constructs (JPG, 62.5 KB)
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(A) Lysates from self renewing WT-GFP, WT-Stat5a-ER* and WT-cS5-ER* cultures were analyzed for P-Akt and total Akt. eIF4E, loading control. (B) 293T cells were transiently transfected with Stat5a-ER*, cS5-ER* or GFP constructs, together with murine EpoR cDNA. 48 hours later, cells were left untreated or treated for 30min with Epo, 4-OH-T, or Epo plus 4-OH-T. Extracts were analyzed for DNA-binding of transfected Stat5 constructs to a beta-casein-specific promoter sequence by EMSAs.
- Figure S4. Jak2−/− cells expressing cS5 can undergo myeloid differentiation in vivo (JPG, 105 KB)
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Equal numbers of cS5 expressing E12.5 WT- (left panels) and Jak2−/− fetal liver cells (right panels) were injected into sub-lethally irradiated mice. Peripheral blood of engrafted animals was monitored for GFP-positive myeloid cells 6 months post-transplant. FACS plots for GR-1 and GFP (top) and Mac-1 and GFP (bottom), of a typical transplant individual from each group are shown.
- Figure S5. Working models how activation of Stat5 allows erythropoiesis in the absence of Epo, EpoR, and Jak2 and how cytokine receptors and receptor tyrosine kinases such as the EpoR or c-Kit could cooperate in activating Stat5 via Jak2 (JPG, 103 KB)
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(A) In WT cells, erythropoiesis requires functional Epo signaling through EpoR/Jak2, leading to tyrosine-phosphorylated, transcriptionally active Stat5 (left). Expression of the hyper-activatable Stat5 mutant cS5 allows erythropoiesis in EpoR−/− and Jak2−/− cells and abrogates Epo dependence of erythropoiesis (middle), arguing for involvement of another kinase X, possibly a member of the Jak family (data not shown). Alternatively, 4-OH-T induced dimerization and activation of Stat5-ER* fusion constructs allows erythropoiesis in the absence of Epo signal transduction (right). This indicates that tyrosine phosphorylated, DNA-bound, transcriptionally active Stat5 dimers (i.e. Epo-activated endogenous Stat5, cS5 or 4-OHT-induced Stat5a-ER*) are sufficient to enable erythropoiesis. (B) Functional interactions between cytokine receptors and receptor tyrosine kinases are likely to occur mainly in immature hematopoietic cells, co-expressing cytokine receptors and growth factor receptor tyrosine kinases. Other pairs of cytokine receptors and receptor tyrosine kinases (gray) might cooperate similarly, via Jak2 and/or other Jaks.
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