Figure 2.

Disruption of TLR4 suppresses cooperative effect of stearic acid with TNF-α for MIP-1α/CCL3 production. (A,B) THP-1 cells were incubated with oxidized 1-palmitoyl-2-arachidonyl-sn- glycero-3-phosphorylcholine (OXPAPC) (30 µg/mL) for 1 h and then treated with stearic acid and TNF-α for 24 h. Cells and culture media were collected. MIP-1α/CCL3 gene expression was determined by real time PCR and secreted MIP-1α/CCL3 protein was determined in culture media by ELISA. (C,D). Monocytic cells were treated with 2 µg/mL of neutralizing TLR4 mAb or isotype-matched control (IgA2) for 40 min. Antibody-treated cells were treated with stearic acid and TNF-α for 24 h. MIP-1α/CCL3 mRNA and secreted MIP-1α/CCL3 protein were determined. (E–G) Monocytic cells were transfected with either control or TLR4 siRNA. TLR4-deficient cells were stimulated with stearic acid and TNF-α for 24 h. Knockdown efficiency of transfection was checked by TLR4 gene expression with qRT-PCR. MIP-1α/CCL3 mRNA and secreted MIP-1α/CCL3 protein were determined. The results obtained from minimum three independent experiments with three replicates of each experiment are shown. All data are expressed as mean ± SEM (n ≥ 3); * P < 0.05; ** P < 0.01when compared with Stearic acid or TNF-α alone.