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Blood, Vol. 113, Issue 5, 1027-1036, January 29, 2009
SHD1 is a novel cytokine-inducible, negative feedback regulator of STAT5-dependent transcription
Blood Nakajima et al.
113: 1027
Supplemental materials for: Nakajima et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 58.9 KB)
- Table S1. Cellularity of spleen, thymus, and bone marrow from wild-type or SHD1-mutant mice (PDF, 170 KB)
- Table S2. Colony assay for hematopietic progenitors in the bone marrow (PDF, 78.2 KB)
- Table S3. Primer sequences for RT-PCR (PDF, 369 KB)
- Figure S1 The effect of SHD1 on non-STAT5 promoters (JPG, 47.1 KB)
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293T cells were transfected with reporter construct responsive either to STAT1 (A), STAT3 (B), SMAD3 (C), or NFκB (D). Increasing amount of SHD1 expression vector was co-transfected to examine the repressive effect of SHD1. The data are mean +/− S.E. (n=3).
- Figure S2. Flow cytometric analysis of bone marrow cells (JPG, 195 KB)
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Bone marrow cells from wild-type, SHD1+∕−, or SHD1−∕− mice were stained with antibodies labeled with FITC or PE, and then were analyzed by flow cytometry.
- Figure S3. Flow cytometric analysis of spleen cells (JPG, 119 KB)
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Spleen cells from wild-type, SHD1+∕−, or SHD1−∕− mice were stained with antibodies labeled with FITC or PE, and then were analyzed by flow cytometry. The percentages of cells in each quadrant are shown on the right of each panel. UL, upper left; UR, upper right; LL, lower left; LR, lower right.
- Figure S4. Flow cytometric analysis of thymocytes (JPG, 46 KB)
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Thymocytes from wild-type, SHD1+∕−, or SHD1−∕− mice were stained with the antibodies labeled with FITC or PE, and were analyzed by flow cytometry. The percentages of cells in each quadrant are shown below each panel. UL, upper left; UR, upper right; LL, lower left; LR, lower right.
- Figure S5. Induction of IL-2 receptor αchain in wild-type or SHD1 mutant mice (JPG, 61.9 KB)
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Purified splenic T cells were stimulated with anti-CD3 for 2 days, and the surface expression of IL-2 receptor α chain was analyzed by flow cytometry.
- Figure S6. Th1/Th2 differentiation of wild-type or SHD1 mutant mice (JPG, 53 KB)
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Purified splenic T cells were examined for their ability to differentiate into Th1 or Th2 cells. To examine the in vitro development of the Th1 and Th2 cells, two-step culture was employed. First, purified splenic CD4+ T cells were cultured for 6 days with plate-coated anti-CD3 in the presence of soluble anti-CD28. For the second step culture, proliferated cells were re-stimulated with plate-coated anti-CD3 after the first-step culture. One day after stimulation, IFN-γ– and IL-4–producing cells were determined by intracellular cytokine staining.
- Figure S7. Induction of STAT1- or STAT3-responsive gene in SHD1 mutant mice (JPG, 63.4 KB)
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Mouse embryonic fibroblast (MEF) prepared from SHD1-mutant mice were stimulated with indicated concentrations of IFNγ (A) or IL-6 + sIL-6R (B) for the times indicated. Cells were harvested and subjected to quantitative real-time RT-PCR analysis for IRF-1 (A) or SOCS-3 (B) expression. Data are mean +∕− S.E. (n=3).
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