The role of TLR4 in PS-F2-induced macrophage activation. (A) BMDMs from C3H/HeN (wild type) and C3H/HeJ (TLR4 mutant) mice were pre-incubated with or without laminarin (0.3 mg/ml) or piceatannol (25 μM) for 1 h and stimulated with PS-F2 in the presence or absence of laminarin or piceatannol for 20 h. BMDMs left untreated or stimulated with poly (I:C) (10 μg/ml) served as controls. TNF-α concentrations in the culture fluids were determined by ELISA (n = 3). (B) RAW264.7 cells were stimulated with LPS (0.5 μg/ml) or PS-F2 (15 μg/ml) for 5 h (first stimulation), and after medium removal and washing, fresh media containing LPS (0.5 μg/ml) or PS-F2 (15 μg/ml) were added as indicated (second stimulation). At 20 h after second stimulation, TNF-α concentrations in the culture fluids were determined by ELISA (n = 3). Data shown are representative of 3 or more experiments. **P < 0.01.