Notch1 is highly activated in human SCLL cells. (A) Flow cytometric analyses of PB from the 8;13 EMS showing a CD4⁺/CD8⁺ immunophenotype, with most cells expressing both CD4 and CD8 at intermediate levels (top left). Fewer TCRα⁺ and TCRβ⁺ cells (top right) were seen, as well as an increase in myeloid (Mac1⁺/CD11c⁺) cells (bottom left). Flow analysis using a Notch1 antibody labeled with RPE (AbD Serotec, clone mN1A) showing high levels of intracellular Notch1 in both the human 8;13 cells and Jurkat cells compared with the normal PB sample (bottom right). (B) Western blot analysis of KG-1 cells harboring FGFR1OP2-FGFR1 (top). Flow analysis showed higher levels of cellular Notch1 compared with normal human PB (Nor. PB) and 3 BCR-ABL–positive CML samples. Jurkat cells were used as a positive control for high Notch1 expression (middle). Western blot analysis using the V1744 antibody (bottom) demonstrates the activated form of Notch1 in KG-1 and Jurkat cells but not in normal or BCR-ABL–positive cells (bottom). KG-1 cells treated with either DAPT (5μM) or Comp E (5μM) show decreased activated Notch1. (C) KG-1 cells express higher CD25 levels on the cell surface (top) as well as Hes1 gene expression (bottom) compared with K562 cells. (D) GSI DAPT can remarkably inhibit KG-1 cell growth (right), whereas K562 cells are relatively resistant to the drug treatment.