Macrophage activation induced by rMRP14.

<p>(a) Increase of TNF-α secretion dependent on rMRP14 concentration in RAW264.7 cells. RAW264.7 cells (2 x 10⁵ cells/ml) were stimulated by rMRP14 (0.15–5 µg/ml) and the concentration of secreted TNF-α in supernatant was measured by ELISA. (b) The increase of TNF-α by rMRP14 could not be blocked by addition of polymyxin B. RAW264.7 cells (2 x 10⁵ cells/ml) were stimulated by rMRP14 (100 ng/ml), EGFP (100 ng/ml) or LPS (2.5 ng/ml) incubated with or without polymyxin B (50 μg/ml), which is a LPS inhibitor. EGFP is recombinant proteins with polyhistidine-tag, which was used as a negative control. (c) MRP14 as an agonist of TLR2 and TLR4. HEK293 cells transfected with murine TLR gene were stimulated by rMRP14 (5 µg/ml) and SEAP induced by NF-<i>κ</i>B activation through TLR was measured. Different letters on the bars represent statistical difference between groups. (d) mRNA expression of IL-1β, TNF-α, IL-6, CCL2, CCR2, iNOS and β-actin in RAW264.7 cells (2 x 10⁵ cells/ml) stimulated by rMRP14 (5 µg/ml). Graphs show mean and SD of triplicates. Data are representative of three independent experiments. ^*<i>P</i> < 0.05; n.s., not significant.