Effect of Nutlin-3 on Notch1 mRNA expression levels in leukemic cell lines. B-lymphoid (BJAB and SKW6.4) and myeloid (HL-60 and OCI) leukemic cells were either left untreated or exposed for 24 hours to Nutlin-3 (10 μM). Levels of Notch1 mRNA and protein were analyzed by quantitative RT-PCR (A) and Western blot analysis (B), respectively. (A) For each cell line, after normalization to the level of GAPDH mRNA, results were expressed as fold of Notch1 mRNA induction in Nutlin-3–treated cultures with respect to the control untreated cultures. Data are reported as means ± SD of results from 3 to 5 independent experiments each performed in duplicate. (B) The levels of Notch1 and of p53 proteins were assessed by Western blot analysis in cell lysates. Tubulin staining is shown as a loading control. Representative examples of Western blot results of 5 independent experiments are shown. After densitometric analyses, results were expressed as fold of Notch1 protein induction in Nutlin-3–treated cultures with respect to the control untreated cultures. Data are reported as means plus or minus SD of results from 5 independent experiments. *P < .05 with respect to untreated cells.