AML-induced NOX2-derived superoxide drives BM senescence. (A) BMSC from p16-3MR were isolated and then cultured alone or with lin⁻, MN1, or HoxA9/Meis1 cells for 3 days. DCF fluorescence was assessed in BMSC by flow cytometry (n = 4). (B) Human BMSC were treated with 10 μM H₂O₂ for 6 days and then analyzed for senescence associated SA-βgal, and (C) p16 mRNA expression (n = 4). (D) C57BL/6 mice injected with MN1. At 21 days postengraftment, mice were euthanized and whole BM was isolated and analyzed for H₂O₂ production using the Amplex red assay (n = 5). (E) C57BL/6 mice were injected with MN1 cells. At 21 days postengraftment, mice were euthanized and BMSC were analyzed by flow cytometry for DCF fluorescence (n = 5). (F) Real-time PCR assay was used to analyze the NOX2 mRNA expression level in NOX2-KD MN1 cells compared with control-KD cells (n = 4). (G) Kaplan-Meier survival curves for p16-3MR mice injected with MN1 NOX2-KD cells or MN1 control-KD cells (n = 7 in each group). (H) At the end point of the experiment, BM was isolated and flow cytometry was performed to detect BMSC-derived RFP (n = 5). (I) Kaplan-Meier survival curves for p16-3MR mice injected with MN1 NOX2-KD cells or MN1 control-KD cells and then injected IP with PBS or GCV at day 15, GCV (25 mg/kg) for 5 days (n = 4).