Excessive ROS in bystander human hematopoietic cells results in DNA damage in HSPCs. (A) Flow cytometric analysis of fold change of ROS levels by DCF-DA staining in human CD34⁺ cells of in vitro RIBE model. (B) Gene set enrichment analysis of the activation of the p53 signaling pathway, negative regulation of the cell cycle, and positive regulation of the apoptotic signaling pathway in human CD34⁺ cells of the in vitro RIBE model. (C) Real-time quantitative polymerase chain reaction analysis of the related genes in human CD34⁺ cells of in vitro RIBE model. (D) Western blot verified the expression of related signaling pathway at the protein levels. Each bar represents the mean ± standard deviation for biological triplicate experiments. (E) Human CD34⁺ cells of in vitro RIBE model were immunostained for p-p53, γ-H2AX, p-ATM, p-53BP1, and FOXO3a (p-p53, γ-H2AX, p-ATM, p-53BP1, and FOXO3a, green; 4′,6-diamidino-2-phenylindole, blue). Scatter plots represent foci per cell from each group (scale bars, 7 μm). (F) Flow cytometric analysis of fold change of ROS levels by DCF-DA staining of human CD34⁺ cells after different processing times. (G) γ-H2AX expression of CD34⁺ cells at the protein level after different processing times. (H) Relative RNA expression of cytokines of nonirradiated or irradiated human bone marrow cells (n = 3 per group). (I) Human cytokine array showed the relative expression of IL-1ra in nonirradiated or irradiated bone marrow supernatant; the right panel shows the quantification results. (J) γ-H2AX expression of CD34⁺ cells at the protein level in different cytokine-treated groups (*P < .05; **P < .01; ***P < .001).