EGFP⁺CD4⁺CD8⁺DP cells at the early phase of ICN1-induced leukemia overexpress TCR-β at the cell surface and present enhanced expression of Notch-signaling-related genes. (A) Three BALB/c wt mice underwent transplantation with ICN1-transduced bone marrow cells. Two weeks after BMT, cells were harvested, pooled, and sorted, as indicated by the square marker, to obtain purified EGFP⁺CD4⁺CD8⁺ cells from bone marrow and spleen of mICN1 donors (BM-mICN1 and SPL-mICN1, respectively). DP thymic cells sorted from sex- and age-matched wt mice were used as controls (THY WT). (B) Cells sorted as in panel A were stained for TCRβ expression at the cell surface: number indicates the percentage of TCRβ⁺ cells; data are representative of at least 3 independent experiments. (C) Semiquantitative RT-PCR of Notch-signaling-related genes (endogenous Notch1, as eNotch1; Notch3; Deltex1, and Ptcra as pTα) was performed on serial 1:5 dilutions of cDNA derived from cell lysates of pools sorted as in panel A. Numbers indicate different pools derived from each population by 2 independent experiments. PCR were normalized according to β-actin expression.