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Blood, Vol. 113, Issue 12, 2661-2672, March 19, 2009
High GATA-2 expression inhibits human hematopoietic stem and progenitor cell function by effects on cell cycle
Blood Tipping et al.
113: 2661
Supplemental materials for: Tipping et al
Electrophoretic mobility shift assay
Bandshift experiments were essentially performed as previously described.26 Briefly, nuclear extracts were added to a mix of 32P-dCTP–labelled GATA oligonucleotide in binding buffer (20mM HEPES, 50mM NaCl, 1mM MgCl2, 4% Ficoll). Anti–GATA-2 antibody (sc9008, Santa Cruz) was added to these assembled mixes where appropriate. Mixes were run on 4% acrylamide (19:1) gels in 0.25 × TBE at 250V before drying and autoradiography. Transplantation assays
All animal studies were approved by our institutional review board and animals maintained in accordance with local regulations. For 8-week engraftment analyses, sorted, transduced and washed CD34+CD38− cells were injected into the tail-vein of sublethally-irradiated (350 cGy) NOD/SCID mice. Each mouse received around 50,000 cells admixed with untransduced CD34+CD38+ cells and 1 × 106 irradiated CD34-depleted accessory CB cells. Aliquots of transduced cells were maintained in culture for 72hrs for assessment of GFP expression. Small bone marrow aspirates were taken at 4 weeks, and animals sacrificed at 8 weeks. For later experiments, transduced and washed CD34+ cells were injected directly into the bone cavity of anesthetized animals (~3.8 × 105 each). For all transplanted animals, both tibias and femurs were flushed with PBS + 5% FCS, red blood cells lysed with Gey’s solution, and cells stained with anti-human CD45-APC, anti–CD19-PeCy7 and anti–CD33-PE (Becton-Dickinson) for flow cytometry using gates based on unstained engrafted controls and stained unengrafted samples.
Files in this Data Supplement:
- Figure S1 (JPG, 876 KB)
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(A) Growth suppression of CB CD34+ cells does not confer reduced Pyronin Y staining similarly to enforced GATA-2 expression. The first two panels re-present data shown in Fig. 2I, together with Hoechst/Pyronin Y staining patterns of CB CD34+ cells treated with 5ng/mL TGFβ1, 50 µM Olomoucine, 20 µM Roscovitine or 18Gy of γ-irradiation. All cells were treated for 18 hours in culture in the presence of SCF, TPO and Flt3L, then fixed in ethanol and stained with Hoechst and Pyronin Y. Only cells deprived of cytokines show a marked decrease in Pyronin Y staining similar to that observed with enforced expression of GATA-2. (B) Enforced GATA-2 expression confers increased quiescence on CD34+CD38+ cells. Hoechst 33342 and Pyronin Y staining profiles of CB CD34+CD38+ cells from a single representative experiment are shown, as shown in Fig. 2F. Briefly, samples were split and infected with lentiviral vectors, and sorted 3 days later on CD34 and CD38 before staining with Hoechst and Pyronin Y. Hoechst 33342 and Pyronin Y staining profiles are shown for the indicated populations from a representative sample. The mean percentage of quiescent CD34+CD38+ cells is shown in Fig. 2H. (C) Engraftment and in vivo reconstitution of NOD/SCID animals by CD34+CD38− CB cells is inhibited by enforced GATA-2 expression in a level-dependent manner. Staining for human CD45 and GFP signal are shown for all engrafted animals from the Vector-transduced and GATA-2¬–transduced groups at 4 and 8 weeks after transplantation (aspirate samples at 4 weeks are shown next to samples from the same animal sacrificed at 8 weeks).
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