Fig. 3

Dissection of the influence of FCS upon TLR2 or TLR4 mediated pathogen recognition. a TLR2, but not TLR4 signalling activates immune gene expression irrespective of FCS presence. pbMEC were challenges in SM10 or SM0 with synthetic lipopetides (Pam2CSK4, 10 ng/ml; Pam3CSK4, 100 ng/ml) as TLR2 ligands or purified LPS (10 ng/ml) as TLR4 ligand. Same experimental setting as in Fig. 1. The mRNA concentration (ordinate) of TNF or NOS2A was measured at different times after challenge (abscissa). Data are mean values and SEM from two biological replica experiments, each assayed in duplicate; *, p < 0.05 vs. unstimulated control (t-test, with Bonferroni’s correction). b Purified PAMPS activate NF-κB in pbMEC irrespective of FCS supplementation. pbMEC were transfected with the NF-κB reporter expressing vector (same experimental setting as in Fig. 2a) and challenged for 24 h with purified LPS or Pam2CSK4 (10 ng/ml each) or 100 ng/ml of Pam3CSK4. Mean values of increased luciferase activity over that of unstimulated controls (ordinate) from two (LPS, Pam2CSK4) independent experiments, each assayed in triplicate. Different superscript letters indicate significant (t-test, p < 0.05) difference from each other and from the unstimulated control. c Lysates from shattered S. aureus do not strongly activate NF-κB in pbMEC. Same as b, but comparing in pbMEC the NF-κB activation through heat killed bacteria (particles) with that caused by supernatants (lysates) from RiboLyser crushed heat killed pathogens from S. aureus strains N305 and RF122 to that of E. coli. Protein concentrations applied for the respective challenge are given below the columns. Each condition was assayed in triplicate. Superscript letters (a, b) indicate significant (p < 0.05) difference between the column means and from the unstimulated controls