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Blood, Vol. 109, Issue 5, 1887-1896, March 1, 2007
ATM deficiency disrupts Tcra locus integrity and the maturation of CD4+CD8+ thymocytes
Blood Matei et al.
109: 1887
Supplemental materials for: Matei et al, Vol 109, Issue 4, 1887-1896
Files in this Data Supplement:
- Table S1. TcraC and TcraV iFISH results for total thymocytes (PDF, 19 KB) -
An overall test of the equality of distributions for the wild-type versus the mutant total thymocytes was performed using a standard chi-square test for contingency tables. For both the C and the V data, the P values were < .001, providing strong evidence of a difference in the distributions between the wild type and mutants. Note that the C = 0 category was omitted because thymocytes lacking both TcraC signals were not found in either Atm+/+ or Atm−/− thymocytes.
- Table S2. TcraC and TcraV iFISH results for DP thymocytes (PDF, 19.1 KB) -
An overall test of the equality of distributions for the wild-type versus the mutant DP thymocytes was performed as described for Table S1. For the C data the P value was < .001, and for the V data the P value was < .001, providing strong evidence of a difference in the distributions between the wild type and mutants. Note that the proportion of ATM-deficient DP thymocytes with V = 0 signals (19.5%) was different from the proportion of wild-type DP thymocytes V = 0 (1.6%), P < .001.
- Figure S1. Increased apoptosis in Atm−/− cortical thymocytes (PDF, 38.3 KB) -
(A) Atm−/− thymi contain elevated levels of apoptotic cells expressing active caspase-3. Apoptotic cells were revealed by staining for active (cleaved) caspase-3. Immunohistochemistry was performed on 5-µm sections of paraffin-embedded thymi from 4-week-old Atm−/− mice and controls. Panels (80× magnification): (i) Atm+/+ thymic cortex; (ii) Atm−/− thymic cortex; (iii) Atm+/+ thymic medulla; (iv) Atm−/− thymic medulla. (B) The graphs represent the average number of cleaved caspase-3–positive cells per 10 fields (imaged at 20× magnification) in the thymic cortex and medulla of 4-week-old Atm−/− mice and age-matched controls. The numbers of apoptotic cells in the ATM-deficient thymi were significantly different from those in the wild-type thymi (P < .001) by 1-way ANOVA. Each histogram represents an individual animal.
- Figure S2. Overexpression of BCL-2 does not restore generation of TCRint DP or TCRhi SP thymocytes in ATM-deficient mice (PDF, 26.5 KB) -
Percentages and absolute numbers of thymocyte subsets in Atm−/−;Bcl2 Tg+ (gray), Atm−/−;Bcl2 Tg− (white), Atm+/+;Bcl2 Tg+ (hatched), and Atm+/+;Bcl2 Tg− (black) mice. The bar graphs depict the percentages (I) and absolute numbers (II) (mean ± SD) of DN, DP (TCRlow, TCRint, and TCRhi) and CD4 or CD8 SP thymic subsets in 3- to 5-week-old mice. The percentages and absolute numbers of CD8+ SP were determined by gating on the TCRhi CD8+ SP to eliminate ISP cells. The lower panel (III) represents the total thymic cellularity (mean ± SD) as a function of genotype.
- Figure S3. Precise rejoining of Tcra SE recombination intermediates in Atm−/− thymocytes (PDF, 53.7 KB) -
Thymus DNA from Atm−/− and Atm+/+ mice and control nonrearranging tissue (tail) was digested with either EcoRI alone or double digested with EcoRI and ApaLI. The digested DNA was electrophoresed and blotted onto a nylon membrane. The schematics describe the restriction digest used. Precise rejoining of SE generates an episomal circle with a cut site for the ApaLI enzyme.
- Figure S4. Tcra locus abnormalities identified by iFISH (PDF, 33.7 KB) -
Representative iFISH images showing normal (i-ii) and abnormal (iii-vi) nuclei with respect to Tcra locus signals. (i-ii) Diploid complement of Tcra and Tcra signals in thymocyte nuclei from Atm+/+ thymus 3332. (iii) Loss of one Tcra signal in thymocyte nuclei from Atm−/− thymus 3331. (iv) Biallelic loss of Tcra signals in thymocyte nuclei from Atm−/− thymus 3331.
- Document 1. Supplemental materials and methods (PDF, 171 KB)
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