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Blood, Vol. 106, Issue 10, 3396-3404, November 15, 2005
MEK kinase 1 activity is required for definitive erythropoiesis in the mouse fetal liver
Blood Bonnesen et al.
106: 3396
Supplemental materials for: Bonnesen et al, Vol 106, Issue 10, 3396-3404
Files in this Data Supplement:
- Figure S1. Sysmex counter analysis of blood samples (JPG, 48.5 KB)
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Sysmex counter analysis of blood samples from wt, Jnk1–/– and Jnk2–/– (n=4) 3 days after induction of severe anemia. (A) Red blood cell quantity, (B) hematocrit value, (C) hemoglobin concentration, and (D) mean corpuscular volume of erythrocytes are shown.
- Figure S2. Hematoxylin/eosin stained sections of E13.5 placentas (JPG, 145 KB)
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(A) Layer organization of control Jnk2–/– and Mekk1ΔKD Jnk2–/– placentas. The chorionic plate (cp) is indicated. (B) Extensive intermingling of maternal and fetal blood vessels (mostly nucleated erythrocytes) into the spongiothropoblast layer in Jnk2–/– as well as Mekk1ΔKD Jnk2–/– placentas. Scale bars equal 100 µm. Note the reduced number of erythrocytes in fetal blood vessels of Mekk1ΔKD Jnk2–/– placentas, further confirming the anemic phenotype observed.
- Figure S3. Flow cytometry of fetal liver cells from E13.5 wt and Mekk1ΔKD embryos double stained with CD71 and Ter 119 distinguishing various stages of erythroid development (JPG, 136 KB)
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Numbers represent the percentage of cells in each region (R). R1 contains primitive progenitors (including mature BFU-Es and CFU-Es); R2, proerythoroblasts and early basophilic erythroblasts; R3, early and late basophilic erythroblasts; R4, chromatophilic and orthochromatophilic erythroblasts; and R5, late orthocromatophilic erythroblasts and reticulocytes. The dot plots represent the log fluorescence intensities of live cells.
- Figure S4. The numbers of hematopoietic progenitors in E13.5 fetal livers of Jnk2–/– and Mekk1ΔKD Jnk2–/– embryos were determined by standard methylcellulose assay (JPG, 24.3 KB)
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Briefly, for CFU-E and mature BFU-E fetal colonies, liver cells (2 × 105) were plated in duplicate in semisolid methylcellulose (MethoCult 03334; Stem Cell Technologies) and cultured according to manufacturer’s recommendations. Benzidine-positive colonies were counted after 2 and 4 days. For CFU-GMs, CFU-GEMMs, and immature BFU-E colonies, fetal liver cells (2 × 104) were plated in duplicate in methylcellulose (MethoCult 03334; StemCell Technologies) according to manufacturer’s recommendations and scored at day 12. Data represent the number of colonies plus or minus SD per 20,000 total cells, based on duplicate determinations on each of two or three independent fetal livers.
- Figure S5. Peripheral blood was drawn every 2 weeks after adoptive transfer of fetal liver cells from Jnk2–/– or Mekk1ΔKD Jnk2–/– E14.5 embryos into lethally irradiated hosts (JPG, 87.3 KB)
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Samples were analyzed by Sysmex counter analysis for (A) hematocrit value, (B) hemoglobin concentration, and numbers of (C) neutrophils, (D) lymphocytes, (E) monocytes, (F) eosinophils, and (G) platelets.
- Figure S6. Histology revealed no abnormalities in the bone marrow of Mekk1ΔKD Jnk2–/– fetal-liver chimeras (JPG, 96.7 KB)
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Hematoxylin/eosin-stained sections of paraffin-embedded recipient bone marrow, 12 weeks following adoptive transfer of Jnk2–/– (A,C) or Mekk1ΔKD Jnk2–/– (B,D) fetal liver cells. Scale bars equal 100 µm.
- Figure S7. Transmission electron microscopy (TEM) of a fetal liver section from a Mekk1ΔKD embryo, showing an erythroblastic island (JPG, 179 KB)
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A central macrophage (M) containing a nucleus (N) and a phagosome (arrow) is surrounded by erythroblasts (E). Day 13.5 Mekk1ΔKD/+ pregnant mice were fixed by vascular perfusion with 2% glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.2) for 5 min. The uteri were opened and the embryos isolated and stored in the same fixative for 24 hours. Next, the abdominal walls of the embryos were opened and specimens from the liver obtained (embryonic tissues for genotyping were also obtained at this stage). Following three rinses in 0.15 M sodium cacodylate buffer (pH 7.2), the specimens were postfixed in 1% OsO4 in 0.12 M sodium cacodylate buffer (pH 7.2) for 2 hours. The specimens were dehydrated in a graded series of ethanol, transferred to propylene oxide, and embedded in Epon according to standard procedures. Sections were cut with a Reichert-Jung Ultracut E microtome (Leica Microsystems, Nussloch, Germany). Ultrathin sections were collected on one-hole copper grids with Formvar-supporting membranes (Monsanto Chemical, Saint Louis, MO), and were stained with uranyl acetate and lead citrate. The sections were examined using a Philips CM 100 transmission electron microscope (Phillips, Eindhoven, The Netherlands) equipped with a Kodak Slow Scan camera (Kodak, Rochester, NY) and operated at an accelerating voltage of 80 kV. Digital images were analyzed with the analySIS software package (Soft Imaging Systems, Münster, Germany).
- Figure S8. Fixed frozen sections from E13.5 Mekk1ΔKD Jnk2–/– fetal liver were stained for TUNEL (green) for detection of apoptosis followed by staining with PE-conjugated Ter-119 mAb (red) to visualize erythroid cells (JPG, 56.1 KB)
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