1. Growth Retardation and Leaky SCID Phenotype of Ku70-Deficient Mice
Yansong Gu, Katherine J Seidl, Gary A Rathbun, Chengming Zhu, John P Manis, Nienke van der Stoep, Laurie Davidson, Hwei-Ling Cheng, JoAnn M Sekiguchi, Karen Frank, Patricia Stanhope-Baker, Mark S Schlissel, David B Roth, Frederick W Alt 
Immunity 
Volume 7, Issue 5, Pages (November 1997)
DOI: /S (00)

2. Figure 1
Ku70 Protein Deficiency Results in Growth Retardation and Destabilization of the Ku80 Protein
(A) A 4-week-old Ku70−/− (smaller) and its Ku70+/− littermate.
(B) Growth curves of Ku70−/− mice (open symbols), and Ku70+/+ or Ku70+/− littermate controls (filled symbols). Each line tracks the growth of an individual mouse.
(C) Detection of the Ku70 and Ku80 proteins in cell lysates from brain, liver, lung, and kidney of either a Ku70+/− or a Ku70−/− mouse.
Immunity 1997 7, DOI: ( /S (00) )

3. Figure 2
Ku70−/− Cells Become Senescent Earlier than Control Cells and Have Intact Cell Cycle Checkpoints
(A) Growth kinetics of primary MEFs. Passage-4 MEF cells (1 × 105) were cultured and counted at various time points as indicated. Filled squares, Ku70+/+; half-filled squares, Ku70+/−; open squares, Ku70−/−.
(B) Ku70−/− cells accumulate nondividing cells. Asynchronous passage-3 MEF cells were analyzed. The percentages of cells labeled with BrdU in G0/G1 or in G2/M were determined by flow cytometry. Percentage of labeled cells among total live cells is indicated by each box.
(C) Kinetics of Ku70−/− MEF division. Passage-3 synchronized MEF cells were released into complete medium and continuously labeled with BrdU for 24 hr. Symbols as in (A).
(D) DNA damage cell cycle checkpoints and arrest after irradiation. Passage-3 synchronized MEF cells were either irradiated (5 Gy) or nonirradiated and then released into complete medium containing 65 μM BrdU for continuous labeling studies.
Immunity 1997 7, DOI: ( /S (00) )

4. Figure 3 “Leaky” Phenotype in T Lineage Cells in Ku70−/− Mice
(A) Flow cytometry analysis of T cell development in the thymus of 10-day old and an 8-week-old Ku70−/− mice and their littermate controls. (Top) DN, DP, and SP thymocytes and the percentages of these subpopulations gated from total thymocytes using the lymphocyte gate shown (bottom).
(B) Mature T cell subpopulations in lymph nodes of the corresponding Ku70−/− and control mice were stained with CD4, CD8, and TCRβ. The percentages of SP T cells (top) and TCRβ+ T cells (bottom) among total live lymphocytes are shown.
(C) The numbers of total viable thymocytes and cells among the DN, DP, and SP subpopulations. Filled symbols, control mice; open symbols, Ku70−/− mice. Age groups: squares, newborn to 10 days; triangles, 10 days to 8 weeks; and circles, 8–13 weeks.
(D) The numbers of total splenocytes and mature T cell subpopulations. Each symbol represents the same ages of mice analyzed in (C) for thymocytes.
Immunity 1997 7, DOI: ( /S (00) )

5. Figure 4
Heterogeneity of Ku70−/− T Lymphocytes and Development of Thymic Tumors in Ku70−/− Mice
(A) Detection of Vβ8 and Vβ14 in an individual 3-month-old Ku70−/− mouse.
(B) Ku70−/− mice develop thymic tumors. Cells of a thymic tumor in a 3-month-old Ku70−/− mouse and thymocytes from its littermate (Ku70 +/−) were stained with CD4 and TCRβ.
Immunity 1997 7, DOI: ( /S (00) )

6. Figure 5 Impaired B Cell Development in Ku70−/− Mice
(A) Bone marrow cells were analyzed for the presence of pro-B cells (B220+CD43+), pre-B and immature B cells (B220+CD43−), and mature B cells (B220+IgM+).
(B) Absence of B cells in the periphery (spleen), determined by the lack of B220+ cells in the spleen of Ku70−/− mice.
(C) Assay of serum IgM levels by enzyme-linked immunosorbent assay using described methods (Young et al. 1994). Four 8-week-old Ku70+/+ mice (filled circles) were used as controls. Eight 3- to 7-week-old and eight 8- to 14-week-old Ku70−/− mice (open circles) were tested as shown.
Immunity 1997 7, DOI: ( /S (00) )

7. Figure 6 Nucleotide Sequences of Coding Joins and RS Joins
(A) Eighteen sequences of RS joins isolated from pJH200 recombinants in the V(D)J recombination. Deletions are evident by by comparision to full-length template (top). Redundant juctional nucleotides shown at right could be due to homology-mediated joining (HMJ).
(B) Eighteen Dβ1-Jβ1.1 coding joins from Ku70−/− thymocyte diagrammed with respect to full-length D and J sequences (top). Middle, N regions and P elements (none observed); right, center and redundant junctional nucleotides (underlined) indicating the HMJ.
(C) Sequences of four VκJκ coding joins isolated from the spleen of a productive μ heavy chain complemented Ku70−/− mouse. Germline Vκ sequences were unknown, and three of the four joins were in frame.
Immunity 1997 7, DOI: ( /S (00) )

8. Figure 7 Analysis of Rearrangements and Intermediates at the TCR Loci
(A) The TCRδ locus, with Dδ2 and Jδ1 segments, Dδ2Jδ1 coding joins, circular RS joins, and RS ends shown. Boxes, coding segments; filled and open triangles, RSS with 23 and 12 spacers, respectively. Coding and RS joins were detected by PCR assays, and RS ends were detected by LMPCR. Arrows, PCR primers; lines with an asterisk, internal oligonucleotide probes.
(B) Detection of Dδ2 to Jδ1 coding joins in thymocyte DNA. For each sample, 100 ng (H) and 10 ng (L) of DNA was used. The expected size of the rearrangement is 160 bp.
(C) Detection of circular RS joins from serial diluted thymocyte DNA of Ku70+/− and Ku70−/− mice.
(D) Analysis of the fidelity of RS joins by ApaLI restriction digestion. D and U, digested and undigested PCR products, respectively; D with an arrowhead, the smaller fragment generated by the ApaLI digestion.
(E) LMPCR detection of RS ends at the 5′ end of Dδ2. Both D and U products are shown.
(F) LMPCR detection of RS ends at 3′ Jδ1.
(G) Detection of DJ and V(D)J rearrangements at the TCRβ locus. For each sample, 500 ng of Ku70+/−, Ku70−/− (201, 202, 206, and 150), and SCID thymus DNA was serially diluted (1:2) and analyzed for TCRβ rearrangements.
Immunity 1997 7, DOI: ( /S (00) )