Figure 3 : A recombinant protein (Tth) purified from E. coli induces pro-inflammatory mediators in monocytes and activates NF-κB in a TLR4-NF-κB-luciferase reporter gene assay.

From: Biological Activity of Masked Endotoxin

Figure 3

(a) Monocytes were stimulated with different concentrations of a recombinant protein (Tth) expressed in E. coli BL21 Star™ (DE3) or ClearColi® (CC) BL21 (DE3), or with different amounts of LPS (0.1 ng/ml ~1 EU/ml). After 24 h of incubation, supernatants were analysed by ELISA. Single values of two independent experiments are shown. Mean values are indicated by bars. (b) HEK293 cells were transfected with an NF-κB-luciferase reporter plasmid and a plasmid-mix encoding a functional LPS receptor (TLR4, CD14 and MD2). 24 h after transfection, the medium was replaced and cells were stimulated as indicated (LPS: 0.1 ng/ml ~1 EU/ml). After another 24 h, luciferase activity was measured. Relative luminescence values were calculated relative to the untreated controls. Mean values and SD of at least 3 independent experiments are shown. (c) Monocytes were either left untreated (black bars) or treated with 1 μg/ml of a neutralizing IgG monoclonal antibody to human TLR4 for 1 h (gray bars). Thereafter, cells were either not further treated (−) or stimulated with 0.1 ng/ml (~1 EU/ml) LPS or 0.5 μg/ml Tth expressed in E. coli BL21 Star™ (DE3). After 24 h, supernatants were analysed by ELISA. Mean and SD of 7 independent experiments are shown. For statistical analysis, one-way ANOVA with a Tukey post-test was performed. **p < 0.01, ***p < 0.001, n.s. not significant.