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Blood, Vol. 110, Issue 12, 3871-3880, December 1, 2007
Novel role for EKLF in megakaryocyte lineage commitment
Blood Frontelo et al.
110: 3871
Supplemental materials for: Frontelo et al
Files in this Data Supplement:
- Document 1. Supplemental methods (PDF, 67.6 KB)
- Table S1. The sort criteria used to generate the RNA material for the data in Figure 1 is shown, along with the developmental potential of each sorted population based on published cell surface characteristics 35,71-74 (JPG, 89.8 KB)
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- Table S2. EKLF was induced in differentiating EBs by treatment with doxycycline at day 3 or 4 and harvested at day 8 or 9 as indicated (JPG, 43.2 KB)
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FACS analyses focused on hematopoietic progenitor (c-Kit), progenitor/megakaryocyte (CD41), megakaryocyte (CD42b), granulocyte (Gr1), and hematopoietic proliferation (CD71) markers. Percent positive cells are shown. All gates were drawn based on negative controls for each sample.
- Figure S1. Inhibition of megakaryocyte precursor differentiation is dependent on the timing of EKLF induction (JPG, 85.9 KB)
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The morphology of megakaryocyte cultures grown in TPO on OP9 cells is shown. Cells were untreated (no dox) or treated with 1 ug/ml doxycycline at day 4, 6, or 8 (as indicated) and photographed at day 10 after the start of ES cell differentiation.
- Figure S2. EKLF was induced in differentiating EBs by treatment with doxycycline at day 5, harvested at day 6, and subjected to quantitative chromatin immunoprecipitation with an EKLF monoclonal (7B2) or an irrelevant antibody (JPG, 29.7 KB)
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Three regions within the Fli1 promoter used in Figure 7C were monitored: upstream (∼ -2 kB; Fli1A), proximal (∼ −500 bp; Fli1BC), and downstream (∼ +100; Fli1E). The average of triplicates is shown. The data show a 50% enrichment at the positive control  -globin HS2 region, a signal that is likely attenuated due to the fact the cells of interest form only a minority of the total number in the EB. However, using this criteria for positive occupancy, EKLF binding is detected at upstream and proximal, but not downstream, regions of the Fli1 promoter at a slight but significant level. (Binding to Fli1A, Fli1BC and -globin HS2, but not Fli1E, was significant to p<0.05.).
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