Enhanced apoptosis in situ of the DN4 cells from lck/SPA-1 Tg mice. (A) DN4 thymocytes isolated from lck/SPA-1 Tg and SPA-1^flox Tg littermate mice were immediately 2-color stained with PI and annexin V (left), or fixed and stained for DNA with PI (right). (B) Subsets of DN thymocytes were sorted from lck/SPA-1 Tg and littermate control mice, and genomic PCR was done with the use of TCR Dβ and Jβ primers (top left). The DN3 cells were fixed, permealized and stained with anti-TCRβ antibody (top right). Solid line indicates lck/SPA-1Tg; dotted line, SPA-1^flox Tg mice; and fine line, staining with control antibody. RNAs were extracted from DN3 and DN4 cells, and RT-PCR was done with the use of pTα primers (bottom).