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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 S1. Generation of RhoH-deficient mice and Northern blot analysis of RhoH mutant mice (JPG, 110 KB)
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(A) Schematic presentation of the RhoH gene before (wild-type) and after (knockout) homologous recombination of the targeting vector (EcoRI-EcoRI fragment of the knockout gene). Homologous recombinants were identified by digestion of genomic DNA with HindIII and Southern blot hybridization with the indicated probe, resulting in a 6.3-kb fragment for wild-type and an 8.6-kb fragment for the recombined gene. Mice were genotyped using genomic PCR with forward primers specific for wild-type (Rh1) or knockout (Rh4) and a common reverse primer (Rh2). Exons are boxed, and the coding region of RhoH is hatched. neo indicates neomycin resistance expression cassette; A, AatII; H, HindIII; E, EcoRI; ATG, translation start site; TAA, stop codon. (B) Southern blot analysis of ES cell DNA as described in panel A, identifying homologously recombined ES cells. (C) Southern blot analysis of mouse tail DNA as described in panel A, identifying wild type (+/+), heterozygous (+/–), and homozygous (–/–) RhoH mutants. (D) Genomic PCR of mouse tail DNA as described in panel A, identifying wild type (+/+), heterozygous (+/–), and homozygous (–/–) RhoH mutants. Indicated sizes correspond to the expected sizes according to the genomic map. (E) Northern blot analysis was carried out with total RNA isolated from bone marrow (BM), spleen, and thymus of wild-type (+/+), heterozygous (+/–), and homozygous (–/–) RhoH mutant mice. The blot was hybridized sequentially with a probe for RhoH (top), neomycin resistance (neo; medium), and GAPDH (bottom). Transcripts for the endogenous RhoH mRNA (RhoH) and the neo-RhoH fusion transcript (neo-RhoH fusion) are indicated. In heterozygous animals, equal amounts of the neo-RhoH fusion and the endogenous RhoH were detected in all tissues, indicating a 50% decreased expression of the endogenous wild-type RhoH.
- Figure S2. Decreased cellularity of thymus and spleen in the absence of RhoH (JPG, 42.9 KB)
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Cellularity of BM, spleen, thymus, and lymph nodes of 2-month-old RhoH-null and control mice. *P < .05; ***P < .001; n = 5/5.
- Figure S3. Impaired thymocyte development in the absence of RhoH (JPG, 38.8 KB)
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(A) Thymocytes of 6-month-old mice were analyzed for the expression of CD4 and CD8 by FACS. **P < .01; n = 5 of 5. (B) Thymocytes of 6-month-old mice were gated for lineage-negative (B220, CD4, CD8, NK1.1, Mac1, Gr-1, Ter119) cells and analyzed for the expression of CD25 and CD44. DN1 (CD25–CD44+); DN2 (CD25+CD44+); DN3 (CD25+CD44–); DN4 (CD25–CD44–); n = 4/4.
- Figure S4. RhoH is not required for the development of γδT cells (JPG, 118 KB)
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(A) Single-cell suspensions of thymus, spleen, and lymph node (LN) were analyzed by FACS for the expression of TCR  and CD3. Total cell numbers of TCR-expressing cells (TCR+ CD3+) are shown. RhoH-null mice had normal numbers of T cells in thymus and spleen and increased levels in LN. *P < .05; n = 3 of 3. (B) In the absence of RhoH, the amount of TCR-expressing DN4 cells was unaltered, whereas the small population of TCR+ DN3 cells was significantly elevated in RhoH-null mice. Bar graph presents the absolute numbers of TCR-expressing cells in the DN3 and DN4 populations. **P < .01; n = 4 of 3. (C) RhoH-null DN thymocytes showed a significantly increased percentage of CD5low cells. Percentages of cells marked in histograms are shown. **P < .01; n = 2/2.
- Figure S5. RhoH is expressed at all stages of T-cell development (JPG, 47.1 KB)
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Relative expression of RhoH was determined by qRT-PCR of total RNA isolated from sorted DN1, DN2, DN3, DN4, DP, CD4SP, CD8SP, splenic CD4+, CD8+, B220+, and BM Gr-1+ cells, as indicated. RhoH was expressed in thymocytes at all developmental stages, in mature B (B220+) and T (CD4+, CD8+) cells but was only weakly expressed in granulocytes (Gr-1+) and not detectably expressed in keratinocytes (keratin). GAPDH was used as a reference gene to normalize RhoH gene expression. RhoH expression level in DP cells was set to 1. Shown are results of 4 real-time PCR reactions performed with cDNA generated from RNA from cells of 2 independent sorts.
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