Figure 5

Dose–response curves of NF-κB induction by whole bacterial cells and LPS of E. coli K-12 strains. The samples were assayed with HEK-Blue hTLR4 cells for hTLR4/MD-2-mediated NF-κB induction by colorimetric determination of NF-κB-dependent SEAP activity (A and C). HEK-Blue Null2 cells, the parental cell line of HEK-Blue hTLR4 cells lacking the hTLR4/MD-2 receptor complex, were used as a control (B and D). HEK-Blue hTLR4 and Null2 cells were stimulated with tenfold serial dilutions of whole bacterial cells (A and B) and LPS extracts (C and D) of KPM318 and KPM335 in comparison to their parental strain BW30270, respectively. The values represent the means and standard deviations from three individual experiments. In all experiments, assayed samples showed low level stimulation of HEK-Blue Null2 cells, indicating that NF-κB-dependent SEAP expression was specifically induced via the hTLR4/MD-2 signaling pathway in HEK-Blue hTLR4 cells.