NaChl induces apoptosis of Bcr-Abl–positive cells. (A) Flow cytometric analysis of annexin V binding. Cells were left untreated (NT) or were treated with NaChl (10 μg/mL) for 24 hours (T) before analysis. Values in the quadrants represent percent positive cells. (B) DNA cell cycle analysis after treatment of cells with NaChl (10 μg/mL) for 48 hours. Gates were set to assess the percentage of dead (< 2n DNA, M1), G₀/G₁ (2n DNA, M2), and S + G₂ + M(> 2n DNA, M3). Bars denote boundaries of cell cycle phases. (C) Immunoblot analysis of caspase-3 activation. Cells were treated with NaChl (10 μg/mL) for 48 hours, harvested, and lysed by boiling in SDS sample buffer, and an equivalent amount of lysates was separated by SDS-PAGE and electrotransferred. The filters were probed with anti–caspase-3 that recognizes both procaspase-3 (32 kDa) and caspase-3 cleavage product (17 kDa). (D) Confocal fluorescence images of NaChl-treated (10 μg/mL for 24 hours) K562 and Molt 4 cells after staining with annexin V–Alexa. Molt 4 cells were used as negative control. (E) Time kinetics of apoptosis determined by different techniques. K562 cells were treated with NaChl (10 μg/mL) for indicated times and analyzed for annexin V binding, DNA cell cycle (apoptotic nuclei [Sub G₀/G₁ peak]), and cleaved caspase-3 by flow cytometry.