Figure 4.

Thymopoietic recovery is accelerated in the absence of TRIB2 after genotoxic stress. (a) Thymic cellularity of mice after 4 and 14 days of 5-FU treatment. (b) The white arrow indicates representative thymus of the dissected WT and Trib2^−/−mice. (c) The cellularity of non-T-lineage cells (DC, CD11c⁺; NK cells, Nk1.1⁺; B, CD19⁺B220⁺; Myeloid, Gr-1⁺CD11b⁺) in thymus (n=3 per genotype) were determined by flow cytometry. Cellularity for thymic subsets (d) and DN1 subsets (e). (f) PCR analysis (n=3 per genotype) of Tcrb rearrangement involving the D_β2 to J_β2 gene segments. GL denotes the position of the germline PCR product and number indicates the different rearrangements. 1 kb M, 1 kb DNA marker; neg ctrl, no template negative control. (g) Flow cytometry to determine the fraction of developing thymocytes in resting (G₀) and active (G₁–S/G₂/M) cell cycle. Each phase is indicated in the outlined areas (top row). The corresponding values in the representative staining profile of WT (middle row) and Trib2^−/− (bottom row) mice (n=2–3 per genotype) are frequency of each phase and graphed in h. (i) The frequency of BrdU uptake by DN3 thymocytes after 1 and 4 h of pulsing. A representative staining profile of WT and Trib2^−/− mice (n=3 per genotype per studied time point) is shown here. The values indicated in the outlined areas are the frequencies of BrdU⁺ DN3 thymocytes and graphed in j. For a, d and e, n=9 per genotype per studied time point. 5-FU dosage for a–e was 250 mg kg⁻¹, whereas for f–j it was 200 mg kg⁻¹. For statistical analyses, unpaired Student’s t-test was used for a and two-way ANOVA was used for c–e, h and j. *P<0.05; **P<0.01; ^*** P<0.001, all quantified data are presented as mean and s.e.m. ANOVA, analysis of variance.