Figure 4.

Inhibitors of apoptosis abrogate the effects of ADA deficiency in FTOC. FTOCs were performed with bcl-2 transgenic mice (a), Ada^m1/m1 mice (b), or Apaf-1^–/– mice and control littermates at day 15 of gestation. The ADA inhibitor dCF (5 μM) was added to cultures performed with bcl-2 transgenic mice and Apaf-1^–/– mice and control littermates. The pan-caspase inhibitor z-VADfmk or a control peptide, z-YVADfmk, which was demonstrated to be ineffective in abrogating the effects of ADA deficiency in C57BL/6 FTOCs (data not shown), was added at 100 μM to FTOCs performed with Ada^m1/m1 mice. After 2 days, the cultures were harvested and thymocytes were counted and stained with FITC-anti-CD4 plus PE-anti-CD8. Dead cells were excluded by PI staining. The viable cell recoveries per lobe are shown beneath the dot plots. Representative results are shown from two to four independent experiments. In a separate experiment (d), dATP was measured by HPLC in extracts of 30–35 fetal thymic lobes from normal C57BL/6 mice cultured for 2 days under control conditions or in the presence of z-VADfmk (100 μM), dCF (5 μM), or a combination of both. All cultures contained equivalent concentrations of DMSO (0.25%). The data are representative of three independent experiments.