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Blood, Vol. 114, Issue 7, 1355-1365, August 13, 2009
Regulation of Fas-mediated immune homeostasis by an activation-induced protein, Cyclon
Blood Saint Fleur et al.
114: 1355
Supplemental materials for: Saint Fleur et al
Files in this Data Supplement:
- Figure S6. Cyclon transgenic expression fails to suppress splenomegaly and lymphadenopathy in Fas− mice (JPG, 187 KB)
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Numbers of splenocytes (left panel) and inguinal lymph nodes (right panel) from 8-week-old Fas+, Cyclon-Tg Fas+, Fas− and Cyclon-Tg Fas− mice are shown.
- Figure S7. Generation of Cyclon-EGFP knock-in mice (JPG, 358 KB)
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(A) The Cyclon locus on mouse chromosome 19. B: BamH I, E: EcoR I, V: EcoR V, K: Kpn I, H: Hind III, X: Xba I, Xh: Xho I. (B) Structure of the targeting construct used for homologous recombination in ES cells. Triangles represent the positions of the loxP sites. (C) Structure of the targeted allele (EN) with intact Egfp and pgk-Neor genes. (D) Structure of the targeted allele (E) without loxP-flanked pgk-Neor gene. Arrowheads represent the positions of primers used for genomic PCR. Shaded rectangles represent the positions of the probes for Southerns blot analysis. Southern blot analysis of ES clones 1–2/D2 and 2–3/E-1. Genomic DNA from wildtype (WT) or clones was digested with EcoR V, electrophoresed in agarose gel, blotted to nylon membrane, and hybridized with the probe 1 (E) or 2 (F).
- Figure S8. Number of mice generated from Cyclon+∕EN × Cyclon+∕EN matings (JPG, 80.9 KB)
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- Figure S9. Gross phenotype of Cyclon+∕EN mice (JPG, 406 KB)
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(A) Numbers of splenocytes, CD4 (+) T cells and CD8 (+) T cells from WT and Cyclon+∕EN mice. (B) Expression of CD44 on CD4 (+) and CD8 (+) splenic T cells from WT and Cyclon+∕EN mice. (C) Anti-CD3 Ab-induced proliferation of CD4 (+) and CD8 (+) splenic T cells from WT and Cyclon+∕EN mice. Whole splenocytes were stained with 4 µM carboxy-fluorescin diacetate succinimidyl ester (CFSE) in 1× PBS, 0.1% BSA for 10 minutes at 37°C, activated with anti-CD3 Ab (10 µg/ml) for 4 days and stained with PE-conjugated anti-CD4 or anti-CD8 Abs for FACS analysis.
- Figure S10. Fas expression on CD8 (+) T cells from WT and Cyclon+∕EN mice (JPG, 305 KB)
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(A) Surface expression of Fas on activated CD4 (+) T cells from WT and Cyclon+∕EN mice. Left panel: CD8 (+) T cells were gated on splenocytes that were unstained or stained with anti–Fas-PE Ab (left panel). Right panel: CD8 (+) T cells sorted magnetically were activated with anti-CD3 antibody (2 µg/ml) and anti-CD28 antibody (2 µg/ml) for 3 days and unstained or stained with anti-Fas Ab (solid lines). (B) Expression levels of Cyclon mRNA in activated CD8 (+) T cells from WT and Cyclon+∕EN mice.
- Figure S11. CD4 (+) T cells from WT and Cyclon+∕EN mice were activated with anti-CD3 Ab (2 µg/ml) and anti-CD28 Ab (2 µg/ml) for 3 days in the presence of IL-2 (5 ng/ml) and for additional 1 day only in the presence of IL-2 (5 ng/ml) (JPG, 106 KB)
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Whole-cell lysates were prepared from freshly isolated cells (activation 0) or activated cells (activation 1) and subjected to immunoblot analysis.
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