Figure 3

Impact of TLR4 activation by HSP60 on the inflammatory response in microglia in vitro . Primary microglia were stimulated with HSP60 (1 μg/mL), LPS (100 ng/mL), Pam3CysSK4 (Pam, 100 ng/mL), loxoribine (lox, 1 mM), or CpG ODN (CpG, 1 μm) alone or simultaneously with pairwise combinations of the ligands, as indicated. Supernatants were analyzed (A) by TNF-Α ELISA at various time points, as indicated (mean ± SEM of two to four independent experiments run with duplicates) or (B) by flow cytometry-based multiple analyte detection for cytokine/chemokine levels, as indicated (mean ± SEM of three independent experiments run with duplicates) after 12 h. Data in (A) were analyzed by two-way ANOVA with Bonferroni-selected pairs of each individual compound vs. combination of compounds (*HSP60 vs. ligand combination; #Pam vs. ligand combination). P*, # <0.05; P** <0.005; P*** <0.001. Data in (B) were analyzed by ANOVA with Bonferroni-selected pairs of each individual compound vs. combination of compounds, as indicated. P* <0.05; P** <0.005; P*** <0.001; n.d., not detected.