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Blood, Vol. 112, Issue 3, 576-584, August 1, 2008
EKLF restricts megakaryocytic differentiation at the benefit of erythrocytic differentiation
Blood Bouilloux et al.
112: 576
Supplemental materials for: Bouilloux et al
Files in this Data Supplement:
- Table S1. Oligonucleotides used in this study (PDF, 86.4 KB)
- Figure S1. Schematic diagram of the experimental protocol used to investigate the effect of EKLF knockdown on the megakaryocytic differentiation of normal hematopoietic progenitors cells (JPG, 101 KB)
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CD34+ cells purified from human cord blood were grown for 3 days in the presence of TPO, EPO and SCF and infected for 3 hours with lentiviruses encoding either eklf shRNA or control shRNA with scrambled sequence. Immediately following infection, CD36+CD31Med cells were gated and purified by FACS as indicated (B) and reseeded at one cell per well still in the presence of TPO, EPO and SCF. Remaining unsorted cells were reseeded in bulk in the same liquid culture conditions. Phenotypic analyses of infected GFP+ cells in the unsorted population or GFP+ clones derived from the sorted cells were all performed 7 days post-infection (ie day 10 after the beginning of the experiment) as described in Fig. 4. The rationale for gating this particular CD36+CD31Med cell population was based on our previous unpublished observations. Indeed, the population obtained from purified CD34+ cells after 10 days of culture in the presence of SCF, EPO and TPO is mainly composed of erythrocytic and a few percent of megakaryocytic cells. Kinetic studies revealed that when compared to the initial population which is mainly CD36− and CD31Med, mature erythroid cells (GPA+) appeared CD36+ and CD31low while mature megakaryocytic cells (CD41+) appeared CD36high and CD31high. Based on these observations, we expected that CD36+ CD31Med cells (which are included in a gate located at the bifurcation between that of mature erythroid and mature megakaryocytic cells) might be enriched in bipotent progenitors. The high cloning efficiency (between 55 % and 85%) and the presence of mixed erythro-megakaryocytic colonies in the single cell progeny confirmed that this CD36+CD31Med cell population is indeed enriched in bipotent progenitors (Figure 6). In agreement with these results, we found that this CD36+CD31Medcell population is also CD45RA−IL3Rα− like the previously described human MEP population.56
- Figure S2. EKLF knockdown does not affect the subcellular distribution of FLI-1 and GATA-1 in MEL cells (JPG, 91.3 KB)
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4D7 cells were treated for two days with or without doxycycline to knockdown EKLF followed by two days in presence of HMBA to induce their differentiation as described in Figure 3. Nuclear and cytoplasmic fractions were prepared and equal amounts of proteins from both factions were analyzed by western blot using FLI-1 or GATA-1 antibodies. The same membranes were revealed with a Grb2 antibody as a cytoplasmic control.
- Figure S3. DNA binding mutant of EKLF is unable to antagonize FLI-1 induced megakaryocytic gene expression in K562 cells (JPG, 91.1 KB)
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K562 cells were transfected with either empty vectors, FLI-1 alone and FLI-1 with HA-EKLF or HA-EKLF/M-Zn1-3 expression vectors as indicated. 24 h following transfection, transcripts levels of megakaryocytic genes gpIIIa and gpIX were determined by qRT-PCR. (A) Western blot analysis of FLI-1, HA-EKLF/M-Zn1-3 and HA-EKLF proteins. Actin protein is shown as loading control. Stars indicate unspecific binding of the antibodies. (B) Relative levels of gpIIIa and gpIX mRNAs normalized to hprt mRNA and expressed as fold variations compared to control cells transfected with empty vector (lane 1) (mean and standard deviation from 3 independent experiments).
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