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Blood, Vol. 113, Issue 14, 3337-3347, April 2, 2009
ETS2 and ERG promote megakaryopoiesis and synergize with alterations in GATA-1 to immortalize hematopoietic progenitor cells
Blood Stankiewicz and Crispino
113: 3337
Supplemental materials for: Stankiewicz and Crispino
Files in this Data Supplement:
- Table S1. Selected list of the top 50 differentially up and down-regulated genes in ETS2, ERG, and FLI1 expressing Gata1 knockdown fetal liver hematopoietic cells in comparison to MIGR1 infected Gata1 knockdown cells (PDF, 150 KB)
- Table S2. Primer sequences (PDF, 11 KB)
- Figure S1. Western blot for ERG and ETS2 expression in PlatE fibroblasts (JPG, 39.2 KB)
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Expression of ERG and ETS2 from the MIGR1 construct was confirmed by Western blotting in PlatE cells in comparison to the MIGR1 (two different transfections for ERG and ETS2 are shown in comparison to the MIGR1 control).
- Figure S2. FLI promotes megakaryopoiesis when overexpressed in fetal liver progenitors (JPG, 173 KB)
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(A) CD41 expression, (B) DNA content, and (C) CD42 expression of wild-type cells transduced with MIGR1, ERG or FLI1 and cultured for 72 hours are depicted. To the right of each set of flow plots, graphs of mean +∕− standard error for the CD41, DNA content and CD42 expression are depicted. Fold changes marked with an asterisk have p<0.002. D, (E) qRT-PCR analysis of ETS2, ERG, FLI1, and GAPDH expression in lineage-depleted wild-type E13.5 fetal liver hematopoietic progenitors (D) and in (E) megakaryocytes isolated from 72 hour fetal liver cultures after separation on a discontinuous BSA gradient. Note that levels of FLI1 and to a lesser extent ERG are increased during megakaryocyte differentiation. (F) Graph shows percentages of ETS2 and ERG expression in comparison to FLI1 for fetal liver derived BSA enriched megakaryocytes (same cells as in E). Average +∕− SE is shown (n=3). (G) Protein sequence homology between human FLI1 and ERG.
- Figure S3. ERG overexpression in Gata1-knockdown or Gata1s knock-in fetal liver progenitors results in formation of macroscopic colonies (JPG, 62.9 KB)
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Photos of representative dishes of colonies from the third plating for (A) Gata1-knockdown cells transduced with MIGR1, MIGR1-ETS2, or MIGR1-ERG or B) Gata1s knock-in mice transduced with MIGR1, MIGR1-ETS2, MIGR1-ERG, or MIGR1-FLI1 (see Fig. 5B–C). Macroscopic colonies are only visible for cells that express ETS2 or ERG in the Gata1-knockdown cells and for cells only expressing ERG and FLI1 in the Gata1s knock-in cells.
- Figure S4. Heatmaps from microarray for the gene ontology groups (A) cell cycle, (B) leukemia, (C) apoptosis, and (D) cell proliferation (JPG, 215 KB)
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