S100A8 activates ERK1/2 and JNK signaling pathways through RAGE. A, Cells were pretreated with ERK1/2 inhibitors U0126 and PD98059 and the JNK inhibitor SP600125 for 60 min before the application of S100A8 (10 μg/mL). The phosphorylation was measured in a period of 60 min by immunoblotting with antibodies to pERK, ERK, pJNK, and JNK. B, Alamar blue assay at 48 h showing cells treated with U0126 or PD98059 displayed decreased cell proliferation compared with their control cells. ns, P > .05; * P < .05; ** P < .01; ***, P < .001. C, Cells were pretreated with an inhibitor of RAGE, FPS-ZM1 (1 μM), for 2 h before stimulation with S100A8 (10 μg/mL) for 1 h. Phosphorylation of ERK and JNK was measured in cell lysates by immunoblotting. There was a decrease in pERK/ERK and pJNK/JNK in FPS-ZM1-treated THJ-11T and THJ-16T cells as compared to control cells. D, Alamar blue assay at 72 h showing cells treated with FPS-ZM1 displayed decreased cell proliferation as compared to control cells. ns, P > .05; * P < .05; ** P < .01; ****, P < .0001.