Figure 3
Loss of Bak further enhances survival of thymocytes overexpressing Bcl-2. (a) DP thymocytes isolated by FACS were cultured in medium lacking cytokines (untreated), or following exposure to 10 Gy γ-irradiation, or in the presence of the indicated concentrations of etoposide, dexamethasone, PMA or ionomycin. Cell viability was determined by propidium iodide and Annexin V staining followed by flow cytometry. Stimulus-specific viability was calculated relative to viability of untreated cells at each time point (see Materials and Methods). n=4 from two independent experiments; values are mean±S.E.M. Statistical significance (Student’s t-test) is only indicated for Bak−/−BCL-2tg versus BCL-2tg (*P<0.05, **P<0.01, ***P<0.001) and Bak−/− versus WT (#P<0.05, ##P<0.01, ###P<0.001). (b) Mice (6- to 8-week-old males) were injected intraperitoneally with 30 μg CD3ɛ antibody (black bars) or with an Ig isotype-matched control antibody (anti-TCRγ ; white bars) and thymic analysis performed 40 h later. Data are presented as total cellularity (left), number of DP cells (centre) and DP as % of total thymocytes (right). Data represent mean±S.E.M., n=5–8 mice per indicated genotype. *P<0.05, **P<0.01, ***P<0.001, Student’s t-test
