Rearrangement of TCRβ is impaired in ATMKO DN2/3 cells.

(A) Genomic organization of the murine TCRβ locus. (B) A representative immuno-FISH image of an ATMKO DN2/3 cell showing a 53 BP1 focus (red) at one of two TCRβ loci (green). White bar denotes 1.5 micron. (C) Graph shows mean frequency (±SEM) of ATMWT (WT) and ATMKO (KO) DN2/3 cells expressing 53 BP1 foci at the TCRβ locus. 200 cells of each genotype were analyzed in two experiments. (D) Representative 3D-FISH images of two ATMKO DN2/3 cells hybridized with 5′TCRβ (red) and 3′TCRβ probes (white). In left image probes are separated by less than 1 µm at both TCRβ loci; whereas, in right image probes are separated by more than 1 µm at one of two TCRβ loci. (E) Summary of the frequency of ATMWT and KO DN2/3 cells in which the 5′ and 3′TCRβ probes are separated by more than 1 µm one of two TCRβ loci. More than 300 cells of each genotype were collected in two experiments. (F) Vβ-DJβ rearrangement in genomic DNA from ATMWT and KO DN2/3 cells was quantified using real-time PCR and normalized to invariant Cα. This plot is mean Vβ-DJβ rearrangements±SEM of seven ATMWT and KO data sets analyzed in five real-time PCR experiments. P values: Student’s unpaired (C) and paired (F) 1-tailed t-test and Fisher’ exact test (E). *p<0.05; **<0.01, ***p<0.001.