Figure 2

HMGB1-dependent TLR4 activation requires MD2 and CD14 in vitro. (A) HEK293 cells expressing components of the TLR4 signaling complex were stimulated with 5 or 10 µg/mL HMGB1, 100 ng/mL LPS or 10 ng/mL TNF-α. NF-κB activity is expressed as a fold-increase in the inducible form of luciferase. (B) HEK/TLR4 and HEK/TLR4/MD2 cell lines were transfected with AdCD14 and similarly stimulated with LPS (10 ng/mL) or HMGB1 (1 µg/mL) treatment as above, and NF-κB activation was measured (mean ± SEM, *P < 0.05 versus HEK/WT in [A] and versus HEK/TLR4 + AdCD14 in [B]). Results shown are representative of at least three separate repeats in triplicate.