Effect of GSI on spontaneous B-CLL cell apoptosis and Notch1, Notch2, and Hes1 expression. Freshly isolated B-CLL cells were cultured for 24 hours in complete medium with the indicated GSI concentrations or DMSO as control (n = 12). Cell viability was evaluated by trypan blue exclusion (A) and annexin V/PI staining (B). Apoptosis was evaluated by flow cytometric analysis of annexin V/PI–stained cells (B) and hypodiploid nuclei (C,D). Results in panels A and C are presented as the mean plus or minus SD of all 12 patients examined. *P less than .05, **P less than .01 (GSI vs DMSO) according to Student t test. Results in panel B are presented as the percentage of viable (annexin V⁻/PI⁻), early apoptotic (annexin V⁺/PI⁻), late apoptotic (annexin V⁺/PI⁺), and necrotic cells (annexin V⁻/PI⁺). Results in panel D are presented as the percentage of hypodiploid nuclei. (E) The expression of Notch1, Notch2, Hes1, and BAFF was analyzed by Western blot in whole-cell lysates (25 μg for Notch1, Notch2, and Hes1 and 50 μg for BAFF), and protein loading was assessed by reprobing the blots with an anti–β-actin mAb. In panels B, D, and E, the results shown for patient 6 are representative of 12 patients.