Effects of the PI-3K inhibitor LY294002 on imatinib-treated CML and normal CD34⁺ cells. CD34⁺ cells were cultured with low GF, either without inhibitors (C), or in the presence of 1 μM imatinib (I), 2 μM LY294002 (L), or both (L + I). Data plotted are for CML (▪) and normal (▦) cells. Significant differences between treated CD34⁺ cells and untreated controls (paired t tests; ^*P < .05, ^**P < .01, ^***P < .001) and between CML and normal CD34⁺ cells (unpaired t tests; †P < .05, ††P < .01, †††P < .001) are indicated. (A) Growth of CML and normal CFCs. CML (n = 5) and normal (n = 4) cells were cultured as described for 96 hours and then plated in methylcellulose progenitor culture for 14 to 18 days, and CFC frequency was enumerated. The left panel shows colony numbers per 2000 CD34⁺ cells (mean ± SEM). In the right panel, data are presented as the percentage of suppression of CFCs compared with untreated controls. (B) Proliferation of CML and normal CD34⁺ cells in a CFSE-labeling assay. CML (n = 4) and normal (n = 5) CD34⁺ cells were labeled with CFSE and treated for 96 hours and assessed for proliferation by flow cytometry. ModFit software was used to fit the data and generate a proliferation index. In the left panel, the proliferation index (mean ± SEM) is plotted. In the right panel, results are presented as the percentage of suppression of proliferation compared with untreated controls. (C) Apoptosis of CML and normal CD34⁺ cells. CML (n = 7) and normal (n = 4) CD34⁺ cells were treated for 48 hours and assessed for apoptosis by flow cytometry after labeling with annexinV–FITC and 7-AAD. The left panel shows the percentage of apoptotic cells (mean ± SEM). The increase in apoptosis above that of untreated cells (percentage treated - percentage untreated) is shown in the right panel.