S100A9 promotes terminal differentiation of human AML cells through TLR4. (A) Effect of increasing doses of rhS100A8 and rhS100A9 on expression of CD14 in human CB CD34⁺ cells transduced with MLL-AF9. Expression of the surface marker CD14 was evaluated by flow cytometry on transduced cells (EGFP⁺) after stimulation with different doses of rhS100A8 or rhS100A9 for 48 hours. (B) Proliferation of human MLL-AF9⁺ cells stimulated with 20 µg of rhS100A9 or phosphate buffer saline (PBS) in vitro for 5 days. (C) Kinetics of CD14 expression after stimulation of human MLL-AF9⁺ cells with 20 µg of rhS100A9. (D) Cytospins of human MLL-AF9⁺ AML at different time points showing signs of macrophage maturation induced by rhS100A9 (20 µg/ml). Scale bars, 20 µm. (E) Cytospins of nitroblue tetrazolium (NBT)–positive cells counterstained with safranin 0.15% and percentages of NBT-positive cells. Scale bars, 20 µm. (F) OCR/ECAR ratio indicative of bioenergetic profile of human MLL-AF9⁺ AML cells stimulated with rhS100A9. Data are represented as the mean ± standard error of the mean. P value was determined by Student t test. *P < .05, **P < .01, or ***P < .001 from 3 independent experiments.