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Blood, Vol. 112, Issue 2, 308-319, July 15, 2008
Notch-dependent control of myelopoiesis is regulated by fucosylation
Blood Zhou et al.
112: 308
Supplemental materials for: Zhou et al
Files in this Data Supplement:
- Figure S1. Analysis of peripheral blood platelet, marrow megakaryocyte numbers, HSC subpopulations, and B lineage progenitor cells in FX−/− mice (JPG, 51.3 KB)
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Peripheral blood platelet count was determined using an automated hematology counter (HEMAVET 950FRS) on 12- to 16-week-old wild type (n=9), FX−/− mice reared on standard chow (FX−/−, no fucose, n=9), and FX−/− mice reared on fucose-supplement chow (FX−/−, with fucose, n=9) (A). FACS analysis of the bone marrow CD41-positive megakaryocytes (n=6 for each group) (B), LT-HSC (Flt3−LSK), ST-HSC and MPP (Flt3+LSK) populations in the bone marrow (n=9 for each group) (C). FACS analysis of B lineage progenitor compartment including pre-proB (B220+CD43+BP-1−CD24−), proB (B220+CD43+CD24+), preB (B220+CD43−IgM−) and mature B cells (B220+CD43−IgM+) in the bone marrow and spleen (n=5 for each group) (D–E). Data represent mean ± SD, p>0.5 for parameters unless otherwise indicated.
- Figure S2. Analysis of partial restoration of fucosylated glycans on FX−/− cells derived from FX−/− marrow transplanted into a wild type recipient (JPG, 73.6 KB)
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Marrow LSK or peripheral Gr-1(+) cells were obtained from WT mice, from FX−/− mice reared on standard chow (FX−/−, no fucose), or from WT recipients (Ly5.2) transplanted with FX−/− marrow cells (Ly5.1). Expression of fucosylated glycans on FX−/− donor-derived marrow LSK (A) or peripheral blood Gr-1(+) cells (B) was determined by flow cytometry using an E-selectin-IgM chimera or pea lectin (PSA), while gating on FX−/− donor (Ly5.1) cells.
- Figure S3. Flow cytometry analysis of the conversion of CMP to GMP instructed by OP9 cells expressing different Notch ligands (JPG, 111 KB)
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CMP cells were isolated from FX−/−mice maintained on standard chow (FX−/−, no fucose), or from FX−/− mice maintained for at least 4 weeks on fucose-supplemented chow (FX−/−, with fucose), or from WT mice. These CMP cells were co-cultured for 60 hours with OP9 cells bearing no Notch ligand (OP9-control), or with OP9 cells reconstituted with Notch ligands Dll4, or Jagged1, or Jagged 2 (OP9-Dll4, OP9-Jagged1 or OP9-Jagged2). Cultures derived from fucose-supplement FX−/− mice were supplemented with 1 mM fucose in the culture medium (FX−/−, with fucose). The percentages of GMP and MEP generated from CMP were determined using flow cytometry. Data shown are one representative of at least three independent experiments.
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