AML-derived NOX2 drives mitochondrial transfer. (A) Primary AML (n = 11) and BMSC were prestained with MitoTracker green FM for 1 hour and then cultured together before 24-hour treatment with DPI (1 µM). (B) Four AML patient samples were transduced with a lentivirus targeted to NOX2 or control for 72 hours. NOX2 mRNA levels were analyzed by real-time PCR and normalized to GAPDH. (C-F) Four AML patient samples were transduced with a lentivirus targeted to NOX2 or control for 72 hours. KD AML cells and BMSC were prestained with MitoTracker green FM for 1 hour and then cultured together for a further 24 hours before MitoTracker was assayed by flow cytometry. (D) Superoxide production was detected in NOX2, and control KD AML cells by AmplexRED assay. (E) BMSC were cultured with control KD AML or NOX2 KD AML. BMSC were stained for ROS, using H2DCFDA (10 µM), and visualized for ROS by flow cytometry. (F) Primary AML or (G) nonmalignant CD34⁺ were cultured with BMSC and treated with DPI (1 µM) for 72 hours. AML blasts (F) and nonmalignant CD34⁺ cells (G) were stained with Annexin V and analyzed by flow cytometry.