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Blood, Vol. 109, Issue 6, 2346-2355, March 15, 2007
RhoH is important for positive thymocyte selection and T-cell receptor signaling
Blood Dorn et al.
109: 2346
Supplemental materials for: Dorn et al, Vol. 109, Issue 6, 2346-2355
Files in this Data Supplement:
- Figure S6. Decreased expression of maturation markers in RhoH-null splenocytes (JPG, 41.7 KB)
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(A-B) Splenocytes of 4- to 8-week-old mice were analyzed for the expression of CD4, CD8, and CD5 (A; n = 5 of 4), or CD69 (B; n = 7 of 7) by FACS. Percentages of cells marked in histograms are shown. **P < .01; ***P < .001.
- Figure S7. Reduced amounts of naïve peripheral T cells (JPG, 163 KB)
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(A-B) Splenocytes (A) and lymph node cells (B) of 4- to 8-week-old mice were analyzed for the expression of CD4, CD8, CD62L, and CD44 by FACS. In the absence of RhoH, the percentages of naïve cells (CD62LhighCD44low) were decreased, whereas the relative amounts of effector (CD62LlowCD44high) and CD8+ memory cells (CD62LhighCD44high) were increased. Percentages of cells marked in histograms are shown. **P < .01; ***P < .001. (A) n = 7 of 7. (B) n = 4/4.
- Figure S8. Impaired differentiation of RhoH-null DN3 and DN4 cells in vitro (JPG, 111 KB)
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(A) Sorted DN3 cells were cultured for 8 days on OP9-DL1 cells in the presence of IL-7 and Flt3 ligand. Differentiation from DN3 to DN4 was tested by FACS analysis of lineage-negative cells for the expression of CD44 and CD25. RhoH-null cells showed a significantly higher percentage of DN3 and a lower percentage of DN4. FACS staining for CD4 and CD8 revealed significantly increased levels of DN and reduced levels of DP in the absence of RhoH. Cellularity of the RhoH-null cultures was decreased approximately 9-fold, indicating defective expansion in the absence of RhoH. **P < .01; ***P < .001; n = 5 of 6. (B) Sorted DN4 cells were cultured for 8 days on OP9-DL1 cells in the presence of IL-7 and Flt3 ligand. FACS staining for CD4 and CD8 revealed significantly increased levels of DN and reduced levels of DP in the absence of RhoH. Furthermore, cellularity of the RhoH-null cultures was decreased approximately 4-fold, indicating defective expansion in the absence of RhoH. **P < .01; n = 3/3.
- Figure S9. Defective TCR signaling in the absence of RhoH (JPG, 103 KB)
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DP thymocytes of 4- to 8-week-old mutant mice were sorted by FACS or MACS microbeads. TCR signaling was induced as indicated by cross-linking of biotinylated CD3 and CD4 antibodies with streptavidin for 5 minutes (A-E, I), 30 seconds (G), or indicated times (F, H) at 37°C. Total lysates were analyzed by Western blot for ZAP70-P319 and ZAP70 (A), LAT-P195 and LAT (C), PLCγ1-P783 and PLCγ1 (D), Vav2-P and Vav2 (F), and Erk-P and Erk (I). Immunoprecipitation (IP) for LAT (B) and Vav1 (E) was blotted with antiphosphotyrosine antibodies and reprobed with LAT (B) or Vav1 (E). Amounts of active Rac1 and Rac2 were determined by pull-down assays (G, H). Representative examples of the Western blots are shown (quantification in Figure 6).
- Figure S10. Normal adhesion of RhoH-null thymocytes to ICAM-1 and VCAM-1 (JPG, 56.5 KB)
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Relative adhesion of thymocytes from 4- to 8-week-old mice to the immobilized  L 2 ligand ICAM-1 and to the 41 ligand VCAM-1 is shown. Nonspecific interaction was determined in the presence of EDTA. Different extents of integrin activation were achieved by treatment with Mg2+, Mn2+, PMA, Mn2+, and PMA (ICAM-1 EDTA, n = 6/6; Mg, n = 8/8; Mn, n = 8/8; PMA, n = 4 /4; Mn+PMA, n = 8/7 and VCAM-1 EDTA, n = 6/6; Mg, n = 8/7; Mn, n = 8/7; PMA, n = 4/4).
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