Figure 1.

Detection of circulating peptidoglycan using NOD2 transfected cell line and NF-κB-luciferase reporter gene. (a) Activation with muramyl dipeptide (MDP) or peptidoglycan (PGN) from Staphylococcus aureus (S. aureus). Human embryonic kidney (HEK) 293T cells transfected with nucleotide-binding oligomerization domain (NOD) 2 and nuclear factor (NF)-κB luciferase expression plasmids were stimulated with MDP or S. aureus PGN. After six hours of incubation, luciferase activity in cell extracts was measured and expressed as relative light unit (RLU). The figure is the mean ± standard error of the mean (SEM) of three independent experiments performed in triplicates. (b) Sensitivity of NOD2-detection of both Gram-negative and Gram-positive bacterial PGN added in healthy human plasma. HEK293T cells transfected with NOD2 and NF-κB luciferase expression plasmids were stimulated with various concentrations of PGN from S. aureus or Escherichia coli diluted in plasma from healthy controls. The figure is the mean ± SEM of three independent experiments performed in triplicates. (c) NOD2-detection of both purified and non-purified anaerobic Gram-negative and Gram-positive bacterial PGN incubated in healthy human plasma. HEK293T cells transfected with NOD2 and NF-κB luciferase expression plasmids were stimulated with purified or non-purified anaerobic bacterial PGN from Gram-positive (Clostridium clostridioforme) and Gram-negative (Bacteroides thetaiotamicron) diluted in plasma from healthy controls. The figure is the mean ± SEM of three independent experiments performed in triplicates. (d) Positive signals in plasma from septic patients. HEK293T cells transfected with NOD2 and NF-κB luciferase expression plasmids were stimulated with plasma from healthy controls and sepsis patients. As a positive control, MDP was used. The figure is the mean ± SEM of three independent experiments performed in triplicates. (e) Specificity of NOD2 detection of bacterial PGN fragment in comparison with a frame shift mutant of NOD2 (fsNOD2) transfected system. HEK293T cells, transfected with NOD2 or fsNOD2 and NF-κB luciferase expression plasmids, were stimulated with MDP, TNF-α or IL-1β. The figure is the mean ± SEM of three independent experiments performed in triplicates. (f) Comparison of the signals between NOD2 and fsNOD2 in septic plasma. HEK293T cells transfected with NOD2 or fsNOD2 and NF-κB luciferase expression plasmids were stimulated with plasma samples from sepsis patients. The figure is the mean ± SEM of three independent experiments performed in triplicates.