S100A9 activates AML cell differentiation through TLR4, ERK1/2, and c-Jun. (A) TLR4, RAGE, and CD33 expression in human MLL-AF9⁺ cells assessed by flow cytometry. (B) CD14 expression in human MLL-AF9⁺ AML cells. Cells were pretreated with neutralizing human anti-TLR4, human anti-RAGE, or human anti-CD33 (n = 5) and stimulated with rhS100A9 (20 µg/ml) for 48 hours. (C) Phosphorylation of JNK, ERK1/2, p38 MAPK, and transcription factors CREB, c-Jun, and NF-ĸB in Mono-Mac-1 cells (MLL-AF9⁺) treated with rhS100A9 (20 µg/ml) in the presence or absence of antibodies against TLR4. (D) Effect of inhibitors of p38 MAPK (SB352080), JNK (SP600125), and ERK1/2 (U0126) on phosphorylation of transcription factors CREB, NF-ĸB, and c-Jun. (E) CD14 expression in Mono-Mac-1 cells after stimulation with rhS100A9 (20 µg/ml) for 48 hours in the presence of SB352080 (p38 MAPK inhibitor), SP600125 (JNK inhibitor), or U0126 (ERK1/2 inhibitor). Data are expressed 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. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ns, not significant.