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Blood, Vol. 110, Issue 7, 2351-2360, October 1, 2007
Id1, but not Id3, directs long-term repopulating hematopoietic stem-cell maintenance
Blood Perry et al.
110: 2351
Supplemental materials for Perry et al, Vol. 110, Issue 7, 2351-2360
Files in this Data Supplement:
- Document 1. Supplemental methods (PDF, 14.1 KB)
- Figure S1. Id1/GFP expression in response to cytokine stimulation (JPG, 44.6 KB)
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Id1 expression is known to increase in bone marrow when stimulated with myeloid cytokines in culture.3(Doc 1) Accordingly, we tested the responsiveness of our model by culturing bone marrow from Id1GFP/GFP, Id1+/GFP, and wild-type littermates. WBM was collected from mice of the indicated genotypes and suspended in complete Dulbecco modified Eagle medium supplemented with 100 ng/mL stem cell factor, 10 ng/mL interleukin-3, 20 ng/mL interleukin-6, and 10 ng/mL granulocyte colony stimulating factor at 5 × 106 cells/mL. Aliquots of 50 µL were transferred to U-bottomed 96-well microtiter plates and incubated at 37°C for either 3 or 27 hours before analyzing by flow cytometry. Histograms show relative GFP fluorescence intensities at 3 and 27 hours of culture.
- Figure S2. Id1 and GFP protein expression (JPG, 53.3 KB)
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Insertion of the GFP sequence with a stop codon disrupts the Id1 gene and prevents the synthesis of Id1 protein (Fig. 1A). This was verified by immunoblotting cell lysates from cytokine-stimulated bone marrow. After 3 or 27 hours of stimulation, aliquots of cultured marrow from fluorescence studies described for Figure S1 were lysed and immunoblotted with antibodies against Id1 or GFP. Blotting for Erk1 served as loading control. Antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA).
- Figure S3. Hoechst/Pyronin-Y analysis of cell cycle in Thy1.1Lo LSK enriched HSC from wild-type and Id1–/– marrow (JPG, 69.1 KB)
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Cells were stained and sorted as described in methods, then fixed and labeled with Hoechst 33342 and pyronin Y as described by Darzynkiewicz, et al.4(Doc 1) Briefly, 106 sorted cells were fixed by suspending in 10 mL of 70% ethanol overnight at -20°C. Fixed cells were washed in Hank balanced salt solution (HBSS) supplemented with Mg2+ and Ca2+, then suspended at 2 × 106 cells/mL. Immediately before analysis, cells were combined with an equal volume of 2 µg/mL Hoechst 33342 (Invitrogen, Carlsbad, CA) and 4 µg/mL pyronin Y (Polysciences, Inc., Warrington, PA) in HBSS with Mg2+ and Ca2+. Relative fluorescence was measured using a MoFlo cytometer equipped with a UV laser (DakoCytomation, Glostrup, Denmark).
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