PV CD34⁺ cells are more sensitive to treatment with ABT-737 than CB and normal bone marrow CD34⁺ cells. (A) Western blotting demonstrates greater expression of Bcl-xL in PV CD34⁺ cells than CB CD34⁺ cells (CB1, CB2, PV1, and PV2). (B) The flow-cytometric analysis results represent the percentage of apoptotic-positive cells observed after the treatment of CB and PV CD34⁺ cells with ABT-737 at various doses for 3 days. Cells were stained with annexin V. The data were analyzed by the Student t test (*P < .05, **P < .01; n = 6). (C) Effects of 200 and 500 U/mL Peg-IFNα 2a and 250 nM ABT-737 alone and in combination on CB and PV CD34⁺ cell apoptosis. The numbers of apoptotic cells were also determined flow cytometrically after 3 days of incubation. The data were analyzed by the Student t test (*P < .05, **P < .01; n = 6). (D) Western blotting showed that the cleaved forms of caspase-3 were increased in PV CD34⁺ cells after treatment with 500 U/mL Peg-IFNα 2a and 250 nM ABT-737 in combination compared with that observed with Peg-IFNα 2a and ABT-737 alone. The α-tubulin serves as the loading control in the Western blot analysis. (E) Effect of Peg-IFNα 2a and ABT-737 alone or in combination on mitochondrial membrane potential in progenitor cells of PV patients. Cells were stained with the JC-1 mitochondrial membrane potential dye after 2 days of treatment with 200 U/mL Peg-IFNα 2a alone, 250 nM of ABT-737 alone, or a combination of 2 drugs. The cells were observed immediately with a fluoroscope (Nikon Eclipse-E600; 10 × .45 objective lens) using a dual-bandpass filter designed to simultaneously detect fluorescein and Texas Red dyes. In live nonapoptotic cells, JC-1 accumulates as aggregates in the mitochondrial membrane which stain red. In apoptotic cells, JC-1 exists in the monomeric form because of the low mitochondrial membrane potential, staining the cytosol green.