Pharmacologic inhibition of Notch 1 signaling suppresses in vitro growth and in vivo tumorigenesis of ZNF112 cells. (A) GSIs, DAPT, and Comp E significantly inhibit ZNF112 cell growth in vitro at micromolar concentrations. Neither DAPT nor Comp E significantly inhibited normal splenocyte viability at their GI₅₀ dosages. (B) DAPT and Comp E at the GI₅₀ dose inhibited expression of Notch 1 direct target genes, but not Notch1 itself. (C) The ICN (activated Notch1) expression levels were dramatically reduced after treatment with both DAPT and Comp E, as were the Notch3 and Ptera target genes. (D) Flow cytometric analysis showing that both DAPT and Comp E can inhibit cell-surface expression of the CD25 Notch1 target gene in ZNF112 cells (the left peak in each case represents the intrinsic background autofluorescence). (E) Peripheral blood CD4⁺/CD8⁺ and GFP⁺ cells were remarkably decreased after 3 weeks in DAPT-treated mice compared with vehicle-treated mice. (F) Treatment with DAPT can markedly prolong survival of mice transplanted with ZNF112 cells.