Oxidative DDR in human HSCs. (A) CB Lin⁻CD34⁺CD38⁻ cells were cultured for 2 days with or without BSO. Intracellular ROS concentrations were determined by the intensity of DCF-DA staining using a flow cytometer. The relative ROS level is presented as a fold induction compared with the MFI value detected in Lin⁻CD34⁺CD38⁻ cells cultured without BSO. Data were collected from 3 independent experiments (**P < .01). (B) CB Lin⁻CD34⁺CD38⁻ cells cultured with or without BSO were immunostained for γ-H2AX (γ-H2AX: green; DAPI: blue). The number of γ-H2AX foci per cell is shown (right; **P < .01). Bars represent 5 μm. (C) CB Lin⁻CD34⁺CD38⁻ cells treated with BSO were immunostained for ATM (p-S1981: green), 53BP1 (p-S1778: green), CHK2 (p-T68: green), and FOXO3a (green). All bars represent 5 μm. (D) BSO treated or nontreated CB Lin⁻CD34⁺CD38⁻ cells were transplanted into NOG mice. Eight weeks after transplantation, engraftment levels of human hematopoietic cells were assessed by flow cytometry. Data were obtained from a total of 6 recipients in each group (3 independent experiments, **P < .01).