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Blood, Vol. 110, Issue 3, 870-876, August 1, 2007
Disruption of palladin leads to defects in definitive erythropoiesis by interfering with erythroblastic island formation in mouse fetal liver
Blood Liu et al.
110: 870
Supplemental materials for Liu et al, Vol. 110, Issue 3, 870-876
Files in this Data Supplement:
- Figure S1. Unimpaired in vitro hematopoietic colony formation potential of palladin−/− fetal liver cells (JPG, 60.2 KB)
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Single cell suspensions from E13.5 fetal livers were prepared, and a hematopoietic progenitor assay was performed in a 24-well tissue culture plate. To quantitate colonies of erythroid colony forming unit (CFU-E), fetal liver cells were plated in triplicate in 0.6 mL methylcellulose-based medium (MethoCult M3334; Stem Cell Technologies, Vancouver, BC, Canada) containing 150 U/mL recombinant erythropoietin (RD Systems, Minneapolis, MN) at a density of 5 × 103 nucleated fetal liver cells/well. Cells were incubated in a fully humidified atmosphere with 5% carbon dioxide at 37°C. The number of CFU-E colonies was determined after 2 days of culture. Colonies of erythroid burst-forming units (BFU-E) were quantitated by plating 3 × 104 nucleated fetal liver cells in 0.6 mL M3334 (Stem Cell Technologies) supplemented with SCF (50ng/mL, RD Systems), IL-3 (10ng/mL, RD Systems) and counted after 7 days of culture. Colonies of granulocyte-macrophage colony forming units (CFU-GM) were determined by plating 3 × 104 nucleated fetal liver cells in o.6mL M3534 (Stem Cell Technologies), and counted after 7 days. For yolk sac colony formation assay, single cell suspensions from the yolk sacs at E10.5 were obtained by treatment with 0.1% collagenase, the culture conditions for yolk sac derived cells were the same as for fetal liver cells. Figure S1 shows the number of CFU-E, BFU-E and CFU-GM colonies per 104 nucleated fetal liver cells (A) or yolk sac cells (B). Error bar represents plus and minus a SD.
- Figure S2. Macrophages from lethally-irradiated mice can bind palladin−/− erythroblasts as unirradiated control macrophages (JPG, 72.7 KB)
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Six- to 8-week-old wt mice were irradiated in 2 doses of 4.8Gy with an interval of 3 hours. After 96 hours, bone marrow cells were allowed to adhere to coverslip in complete medium (RPMI + 10% fetal bovine serum FBS) for 3 hours, then stripped with PBS lacking calcium and magnesium. Adherent macrophages were allowed to respread in complete medium for 2 hours before receiving fresh E13.5 palladin−/− erythroblasts. Finally, the reconstituted clusters were dipped to remove the unbound cells before being processed for double F4/80-Ter119 immunofluorescence using standard procedures. Unirradiated 6- to 8-week-old wt mice served as positive controls. The images were viewed with an Olympus BX51 microscope (Olympus, Shanghai, China) with an Olympus UPlanFl 40×/0.75 objective, captured with a SPOT RTKE cooled color CCD camera (Diagnostic Instruments, Sterling Heights, MI), and imported into SPOT software (Diagnostic Instruments). FITC (F4/80 positive cells) and PE (Ter119 positive cells) fluorescence are shown. Original magnification × 400.
- Figure S3. Fetal liver gene expression profile after palladin disruption (JPG, 79.7 KB)
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mRNA was extracted from 2 wt or 2 palladin−/− E13.5 fetal livers using Trizol reagent (Invitrogen, Carlsbad, CA) and reverse-transcribed into DNA. Semiquantitative PCR was performed to detect the expression of fetal liver hematopoiesis and apoptosis related genes. There were no differences in the expression of transcription factors required for definitive erythropoiesis (Gata2, Aml1, c-myb); cytokines (Epo, Scf); or apoptosis related genes (IFN-βR, TNF-R1, Bcl-XL, K-ras, EpoR). But the expression of TNF- was significantly upregulated. We observed a significant upregulation of the expression of NF- B family transcription factors RelB and RelC. The expression of primitive globin genes (βhi-globin, Ey-globin) was upregulated, while the expression of definitive globin genes (βmaj+min-globin) was downregulated.
- Figure S4. Cell adhesion and expression of associated cytoskeletal proteins in palladin−/− macrophages are comparable to wt cells (JPG, 58.7 KB)
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Primary cultures of fetal-liver-derived macrophages were generated from E14.5 wt and palladin−/− embryos and cultured on coverslips in DMEM supplemented with 10% FBS plus SCF (50 ng/mL, RD Systems), IL-3 (10ng/mL, RD Systems), recombinant Flt3 ligand (10 ng/mL, RD Systems), recombinant IL-6 (5ng/mL, RD Systems) and GM-CSF (10 ng/mL, RD Systems). (A, B) No significant difference was observed between the brief morphology of (A) wt and (B) palladin−/− macrophages. (C, D) Primary (C) wt and (D) palladin−/− macrophages were fixed and stained with phalloidin-FITC (Sigma, St Louis, MO), F4/80 (Ebioscience, San Diego, CA; cy3-conjugated secondary antibody) and DAPI. (E) Wt and (F) palladin−/− macrophages were fixed and stained with phalloidin-FITC, focal adhesion marker vinculin (Sigma; cy3-conjugated secondary antibody). No significant difference was observed in the staining pattern of phalloidin and vinculin between wt and palladin−/− macrophages. The images in A and B were captured as in Figure S2 The images in C-F were viewed with a Zeiss LSM 510 confocal microscope (Carl Zeiss, Heidelberg, Germany) with a Fluar 20×/0.75 UV objective, captured with a camera equipped by the manufacturer (Carl Zeiss) and processed using Zeiss LSM5 Image Browser software (Carl Zeiss). Original magnification × 100 (A, B) and × 400 (C–F).
- Figure S5. Expression and distribution of adhesion molecules related to erythroblastic island formation in palladin−/− macrophages are identical to wt cells (JPG, 65.8 KB)
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(A,C) Wt and (B,D) palladin−/− fetal liver macrophages were cultured on coverslips as described in Figure S1 for 2 days then fixed and immunostained with integrin-V (Santa Cruz Biotechnology, Santa Cruz, CA; Alexa Fluor 532-conjugated secondary antibody), FITC-F4/80 (eBioscience) and DAPI (A, B) or VCAM-1 (Santa Cruz Biotechnology; Cy3-conjugated secondary antibody), FITC-F4/80 (eBioscience) and DAPI (C, D). No significant difference was observed in the staining pattern of integrin-V and VCAM-1 between wt and palladin−/− macrophages. The images were acquired as in Figure S4. Original magnification × 400 for all panels.
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