Delay of glucocorticoid-induced apoptosis in Puma- or Bim-deficient thymocytes. Animals of the indicated genotypes were injected intraperitoneally with graded doses of dexamethasone and killed 20 hours later. Thymi were harvested, and single-cell suspensions were counted and stained with fluorescence-conjugated antibodies to CD4 and CD8 and analyzed in a flow cytometer. (A) Total thymic cellularity of saline or dexamethasone-injected animals. (B) The percentage of CD4⁺8⁺ double-positive cells and total thymic cellularity were used to calculate (C) the absolute number of immature CD4⁺8⁺ DP thymocytes present in control and dexamethasone-treated animals. Bars represent means ± SE of 3 to 6 animals of each genotype and treatment regimen from at least 3 independent experiments. Statistically significant differences are as follows: (C) CD4⁺8⁺ DP thymocytes: wt versus Puma^-/- (P < .033), wt versus Bim^-/- (P < .05), wt versus vav-BCL2 (P < .013). Thymic sections derived from wt (D), Puma-deficient (E), and Bim-deficient (F) animals 20 hours after exposure to 250 μg dexamethasone were TUNEL stained using FITC-dUTP to detect nicked DNA in apoptotic cells. Nuclei were counterstained using 7-AAD. Sections were analyzed using a ZEISS Axiovert fluorescence microscope. The images are representative of 2 independent stains performed on organs of at least 2 animals per genotype and treatment.