Figure 4.

Massive apoptosis of DN-IkTg CD4SP thymocytes. (A) TUNEL assay of WT and DN-IkTg thymus sections. Upon TUNEL staining, thymus tissue sections were counterstained with hematoxylin and eosin. Histology slides show results from a 93-day-old WT and a 94-day-old DN-IkTg mouse and are representative of 3 independent experiments (original magnification ×10) for a total of 3 WT and 3 DN-IkTg mice. (B) Survival kinetics of DN-IkTg CD4SP thymocytes in vitro. Cell viabilities were determined by propidium iodide exclusion at indicated time points during in vitro culture. Data show the summary of 3 independent experiments with each 1 WT and 2 DN-IkTg mice, respectively. (C) Phenotype analysis of apoptosis-resistant DN-IkTg CD4SP cells. Freshly isolated DN-IkTg thymocytes were incubated in medium for 6 hours and then assessed for cell survival. Propidium iodide staining–negative cells were analyzed for their CD4/CD8 profiles and HSA/TCRβ expression. Contour plots are representative of 2 independent experiments with 3 WT and 4 DN-IkTg mice. (D) Apoptosis pathway gene array analysis of DN-IkTg CD4SP thymocytes. Total RNA from sorted WT or DN-IkTg CD4SP cells were used to probe an apoptosis PCR array for expression of pro- and antiapoptotic genes. Data are representative of 3 independent experiments using sorted CD4SP thymocytes with each 1 WT and 1 DN-IkTg mouse. (E) qRT-PCR analysis of potential Ikaros target gene expression from sorted CD4SP thymocytes. Data are the mean ± SEM of 3 independent experiments with a total of 3 WT and 4 DN-IkTg mice. (F) Intracellular Bcl-2 expression in HSA^hiCD3^hiCD4SP. Freshly isolated DN-IkTg thymocytes were assessed for Bcl-2 expression. The histogram is representative of, and the bar graph displays, the mean ± SEM of 2 independent experiments with each 1 WT and 2 DN-IkTg mouse.