Notch signaling in developing thymocytes inversely affects the expression of HES1 and PTEN. (A) Rag2^−/− E14 FL-derived HPCs cultured with OP9-DL1 cells for 8 days are used to give rise to CD44⁻ CD25⁺ DN3 cells, which are then sorted and returned to either OP9-DL1 or OP9-Ctrl cells for 2 days before analysis. Flow cytometric analysis of CD44, CD25, CD127, and CXCR4 expression is shown for Rag2^−/− DN3 cells cultured for 2 days in the absence (Ctrl) or presence (DL1) of Notch signaling. Overlay histograms showing cell size (FSC), and surface expression of CD127 and CXCR4 of DN3 cells, cultured as indicated, are shown on the right. Data are representative of at least 3 independent experiments. (B) qRT-PCR analysis of mRNA expression, normalized to β-actin, of Notch downstream target genes (Deltex1, Hes1, and c-Myc) and Pten is shown for Rag2^−/− DN3 cells cultured for 1 or 2 days as indicated. Data are representative of at least 3 independent experiments, with standard deviation of the mean shown as error bars. (C) qRT-PCR analysis of mRNA expression (as in panel B) is shown for C57BL/6 ex vivo–isolated DN4 and DP thymocyte subsets. Data are representative of at least 3 independent experiments.