Apoptosis induced by BMI1 down-modulation coincides with elevated ROS accumulation and reduced FOXO3A expression. (A) Transduced CB CD34⁺ cells were cultured in serum-free conditions (HPGM supplemented with SCF and Flt3L) for 10 days, and expansion was monitored. (B) The percentage of apoptotic cells at day 10 was determined by FACS staining for annexin V and PI. (C) Transduced cells were cultured in conditions described in (A), and at day 10 were stained with H2DCFDA to determine the intracellular levels of ROS by FACS. (D) Transduced cells were cultured in the absence or presence of 100 μM NAC for 9 days, after which ROS accumulation was determined by FACS. (E) CFC assays were performed with transduced cells in methylcellulose cultures in the absence or presence of 100 μM NAC. (F) CB CD34⁺ cells were transduced with control (scrambled) scr-RNAi or BMI1-RNAi, and CD34⁺38⁻ GFP⁺ cells were sorted on glass slides. Immunohistochemical staining was performed using antibodies against FOXO3A.