Effects of the MEK-1/2 inhibitor U0126 on imatinib-treated CML and normal CD34⁺ cells. CD34⁺ cells were cultured in the presence of low GF, either without inhibitors (C) or in the presence of 1 μM imatinib (I), 25 μM U0126 (U), or both (U + I). Data plotted are for CML (▪) and normal (▦) cells. Significant differences between imatinib-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) CML and normal CFC growth. CML (n = 7) and normal (n = 6) CD34⁺ cells were cultured as described for 96 hours and 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, CFC 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 = 5) and normal (n = 4) CD34⁺ cells were labeled with CFSE, treated for 96 hours as indicated, and assessed for cell division by flow cytometry as described in the “Patients, materials, and methods.” 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 = 6) CD34⁺ cells were treated for 48 hours and assessed for apoptosis by flow cytometry after labeling with annexin V–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.