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Blood, Vol. 113, Issue 20, 4866-4874, May 14, 2009
The histone acetyl transferase activity of monocytic leukemia zinc finger is critical for the proliferation of hematopoietic precursors
Blood Perez-Campo et al.
113: 4866
Supplemental materials for: Perez-Campo et al
Files in this Data Supplement:
- Figure S6. Differentiation of WT and HAT−∕− MOZ ES cell lines (JPG, 71.1 KB)
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(A) WT and homozygote cell lines for the HAT mutation were differentiated into EBs. Samples from each cell line were harvested at different time points for cell counts and the number of cells generated upon differentiation of 2 × 104 ES cells and days of differentiation are indicated. (B) EBs from WT and homozygote cell lines were photographed under phase contrast optics for comparison at days 1 and 3 of EB differentiation. (C) Flow cytometry analysis of Flk-1 and c-Kit expression on HAT+∕+ and HAT−∕− MOZ EB derived cells. Days of differentiation are indicated. Numbers represent the percent of total population in each fraction.
- Figure S7. Vascular/smooth muscle potential of HAT+∕+ and HAT−∕− MOZ ES cell lines (JPG, 85.5 KB)
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(A) Endothelial colonies generated by Flk-1 positive HAT+∕+ (white bars), HAT+∕− (grey bars), and HAT−∕− (black bars) MOZ cells sorted at different days of differentiation. Bars represent standard error of the mean number of colonies from at least 3 cultures (B) Immunohistochemistry of HAT+∕+ and HAT−∕− cells. Day 4.5 EBs were harvested, and Flk-1 positive cells were purified by FACS sorting and plated on gelatin-coated glass coverslips. Cells were allowed to grow for 10 days and stained for CD31 (PECAM) and SMA. CD31+ cells (endothelial) are shown in red, whereas the SMA (smooth muscle actin) positive cells are green (original magnification, ×200).
- Figure S8. Hematopoietic populations in HAT+∕−, HAT−∕−, and WT EBs (JPG, 67.6 KB)
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(A) CD34+/c-Kit+ cell populations in HAT+∕+, HAT+∕−, and HAT−∕− days 6 and 7 EBs. Single-cell suspensions were analyzed by flow cytometry. (B) Number of macrophage/erythroid (Mac/Ery), primitive erythroid (EryP), definitive erythroid (EryD), multilineage (Mix), granulocyte/macrophage (GM), and macrophage (Mac) colonies generated by CD34+/c-Kit+ sorted fractions from the HAT+∕+ and HAT−∕− day 5 EBs. (C) Representative photographs of CD34+/c-Kit+ cells replating in methylcellulose (magnification 20×).
- Figure S9. Relative expression of MOZ and MORF in HAT+∕+ and HAT−∕− (JPG, 69.5 KB)
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Quantitative PCR for MOZ and MORF (reported to actin level) in HAT+∕+ and HAT−∕− day 14.5 fetal liver (A), 3 months old bone marrow (B) or spleen (C). (HAT+∕+ n=6, HAT−∕− n=7 for fetal liver; HAT+∕+ n=4, HAT−∕− n=4 for spleen and bone marrow).
- Figure S10. Analysis of HAT−∕− and HAT+∕+ ES cells contributions to different tissues of chimaeric mice (JPG, 75.1 KB)
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(A) Wild-type or HAT−∕− ES cells, homozygous for gpi-1b isoenzyme, were injected into C57/Bl6 blastocysts homozygous for the gpi-1a isoform. Injected blastocysts were transferred to pseudopregnant females and born chimaeras were analyzed after weaning. To determine the extent of ES cells contribution to the different tissues, organs were extracted and homogenized and the cells lysed by freezing and thawing. GPI isozyme distributions were determined by electrophoresis on Titan III cellulose acetate plates (Helena Laboratories) in 0.025% Tris-glycine buffer, and staining for GPI enzyme activity.26 BM (bone marrow), PB (peripheral blood).
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