Figure 2

B. pseudomallei LPS signals via TLR4. HEK293 cells were transiently transfected with (A) murine or (B) human TLR2, TLR2/1, TLR2/6, or TLR4; co-receptors CD14 and MD-2; and NF-κB-dependent firefly ELAM luciferase and control β-actin-dependent Renilla luciferase. In (C) HEK293 cells were transiently transfected with human TLR4 and co-receptor CD14 with or without MD-2; and firefly ELAM and Renilla luciferases. Cells were stimulated with media alone, (A & B) Pam3CSK4 1000 ng/mL, E. coli 0111:B4 LPS 10 ng/mL, BP-1 LPS 10 ng/mL, K96243 LPS 10 ng/mL, or BP-1 lipid A 10 ng/mL. NF-κB activation was measured by light emission (relative light units). In (A&B) data plotted are means ± standard deviations of duplicate or triplicate conditions. In (C) the means ± standard deviations of triplicate human TLR4-mediated relative light units (normalized to mean empty vector values) are plotted from parallel experiments with or without co-transfection of MD-2. For (A & B) § indicates p < 0.001 compared with empty vector stimulated with the same ligand, and † indicates p < 0.001 compared with TLR2 stimulated with the same ligand using analysis of variance followed by the Bonferroni post-test. Other comparisons are not shown for clarity. Each graph represents one of two independently performed experiments.