Effects of the NL and RUX combination on CML and normal CD34⁺ cell viability and CFC output. (A) CML CD34⁺ cells (n = 3) were either left UT, or treated with NL (5 µM) or RUX (200 nM) or their combination, and cultured. At 48 hours, apoptosis levels were measured by Annexin-V/7AAD staining. CML CD34⁺ cells (n = 3) were cultured as in (A) for 72 hours before drug washout and plating in methylcellulose progenitor assays. (B) Total CFC output was recorded after 12 days culture and compared with the CFC output for each sample prior to the start of the culture (baseline) (note that the number of colonies following 72 hours culture was adjusted for the expansion of CD34⁺ cells in vitro in each arm relative to baseline). (C) CFC frequency based on their morphology (erythroid-burst forming unit and erythroid-colony forming unit), and (D) granulocyte/macrophage-colony forming unit was also recorded and again compared with baseline. (E) Representative pictures of the size and morphology of recovered CFC in each treatment arm. Normal CD34⁺ cells (n = 3) were either left UT, or treated with NL (5 µM) or RUX (200 nM) or their combination. (F) At 48 hours, apoptosis levels were measured by Annexin-V/7AAD staining. Normal CD34⁺ cells (n = 3) were cultured as in (A) for 72 hours before drug washout and plating in methylcellulose progenitor assays. (G) Total CFC output was recorded after 12 days culture and compared with the CFC output at baseline as explained in (B). All data from independent experiments are presented as mean ± SEM. Significance values: *P < .05; †P < .01; ‡P < .001.