S100A9 induces ROS through NADPH oxidase to activate β-catenin. (A-B) The percentage of (A) ROS positive cells and (B) ROS-MFI assessed by flow cytometry in BM-MNCs isolated from MDS patients (n = 5) and normal donors (n = 2). (C) Representative micrograph (original magnification ×2520, 7.5 µm scale) of β-catenin expression in normal BM-MNCs (n = 3), normal BM-MNCs treated with 5 µg/mL rhS100A9 (n = 3), and MDS BM-MNCs (n = 6) (DAPI, blue; and β-catenin, red; merged images show nuclear β-catenin localization). (D) Quantitation and scoring of confocal images based on the presence of no, low, medium, or high nuclear β-catenin. (E) Wnt/β-catenin target gene expression in WT and S100A9Tg BM cells. (F) Representative micrograph (original magnification ×2520, 7.5 μm scale) of β-catenin expression in WT (n = 5), S100A9Tg (n = 5), and S100A9Tg mice that were treated with ICTA (n = 5) by oral gavage for 8 weeks (DAPI, blue; and β-catenin, red; merged images show nuclear β-catenin localization). (G) Wnt/β-catenin target gene expression in MDS BM-MNCs (n = 4) treated for 48 hours with ICTA. Error bars represent SE. *P < .05 and ***P < .001.