Fig 2: Nucks1 mRNA expression in normal thymi and TLsa. Results from TaqMan qRT-PCR assays to assess Nucks1 expression in normal thymi of Trp53+/-, wild type (WT) and Nucks1+/- mice, and in IR-induced TLs of Trp53+/- and Trp53+/-Nucks1+/- mice. *, P<0.05; **, P<0.01 (for Nucks1 expression in TL of Trp53+/- mice compared to normal thymus of Trp53+/- mice, and for Nucks1 expression in TL of Trp53+/- Nucks1+/- mice compared to normal thymus of Nucks1+/- mice). Note, that 9/13 TLs from Trp53+/- Nucks1+/- mice show significantly increased Nucks1, and 2/13 TLs from Trp53+/- Nucks1+/- mice show significantly decreased Nucks1. b. Results from TaqMan qRT-PCR assays to assess ?Nucks1-ßgeo in normal thymi, and in IR-induced TLs of Trp53+/- Nucks1+/- mice.

Fig 3: Analyses of Dβ-Jβ clonality at Tcrb in IR-induced TLs of Trp53+/− and Trp53+/− Nucks1+/− micea. Representative agarose gels from semi-quantitative PCR analyses to assess Dβ1-Jβ1 and Dβ2-Jβ2 rearrangements at Tcrb using primer pairs P1 and P2 (Dβ1-Jβ1) and P3 and P4 (Dβ2-Jβ2), respectively, as show in (b). Fifty (odd lanes) and 5 ng (even lanes) of genomic DNA isolated from the thymi of normal wild type mice (wt1, wt2) and from IR-induced TLs of mice, with genotypes as indicated, were used. Asterisks: indicate TLs not analyzed by array-CGH. b. Schematic of the mouse Tcrb locus with relative genomic positions of the Vβ segments and Dβ-Jβ-Cβ clusters, and of PCR primers (P1, P2, P3 and P4). c, d. Tcrb rearrangements were also assessed by array-CGH analysis (representative expanded array-CGH profiles for TL-153 and TL-157 are shown). Note: For all TLs with genomic loss at Tcrb, the regions of loss spanned the Vβs and Dβ-Jβ clusters, indicative of Vβ to Jβ1 or Vβ to Jβ2 rearrangements. Minimal genomic loss was found in TL-153, as shown in c., which included Trvb30 segment, and maximal genomic loss was detected in TL-153, as shown in (d), which included the Trvb1 segment.

Fig 4: Immunophenotyping analyses of TL cells isolated from IR-induced TLs of Trp53+/− and Trp53+/− Nucks1+/− micea. Primary TL cells from TLs of Trp53+/− mice were stained for CD4, CD8 and CD3 and analyzed by FACS. Representative 2-parameter dot plots showing that most primary TL cells from Trp53+/− mice were CD4+ CD8+ DP (TL-3) or CD8+ SP (TL-102) with moderate CD3 expression (see also Table 2). b. Same as in (a) for primary TL cells isolated from Trp53+/− Nucks1+/− mice. Note, that large compartments of CD4- CD8- DN TL cells were seen in TLs from Trp53+/− Nucks1+/− mice (TL-180, TL-121 and TL-131; see also Table 2).

Fig 5: Nucks1 is haplo-insufficient in suppressing IR-induced TLa. Agarose gels to show the results from semi-quantitative PCR analysis to detect LOH at Trp53 and Nucks1. Genomic DNAs from tail and TL of the same mouse were used as template. Arrows indicate LOH of the Nucks1 wild type and knockout alleles. M: molecular weight marker; W: wild type allele; K: knockout allele; WT CTRL: tail and thymus of a sham-irradiated 129S1/SvImJ mouse. b. Results from TaqMan qRT-PCR assays to assess Trp53 expression in normal thymi of Trp53+/−, wild type (WT) and Nucks1+/− mice, and in IR-induced TLs of Trp53+/− and Trp53+/−Nucks1+/− mice.