Figure 2.

Mechanistic requirements for the induction of NETosis by low-dose radiation. A and C, NETosis assessment by SYTOX Green staining and confocal microscopy of cultured neutrophils isolated from the blood of healthy human donors (A), heavy-burden patients with cancer (B), or the BM of 4T1 tumor–bearing mice (C) ex vivo treated with 0.5 Gy of γ-radiation and the indicated pharmacologic inhibitors. D, NETosis assessment of healthy donor neutrophils treated with 0.5 Gy of γ-radiation and the indicated doses of antioxidant molecules. E, Human neutrophils were ex vivo treated with 0.5 Gy of γ-radiation, and 0.5 mmol/L of NAC was added or washed from the neutrophil cultures at the indicated time points. Then, cells were incubated for up to 4 hours, and NETs were quantified by SYTOX Green staining. F, ROS production assessments using Amplex red staining of neutrophils treated with the indicated γ-radiation doses over time. Dotted lines represent neutrophils treated with 0.5 mmol/L of NAC in addition to γ-radiation. NETosis assessment on human neutrophils treated with increasing concentrations of NADPH oxidase inhibitor DPI (G) or 0.1 µmol/L DPI when receiving increasing doses of γ-radiation (H). I, NETosis assessments of healthy donor neutrophils treated with 0.5 Gy of γ-radiation and the indicated concentrations of NADPH oxidase inhibitors. J, Human neutrophils were treated with 0.5 Gy of γ-radiation, and 0.1 µmol/L DPI was added or washed from the neutrophil cultures at the indicated time points as shown in (E). Kruskal–Wallis tests followed by post hoc tests were performed for multiple comparisons and Mann–Whitney U-tests for side-by-side comparisons. Differentially colored dots represent individual donors or individual mice.