Annexin A5 reduces LPS-induced activation and maturation of dendritic cells by inhibiting TLR4 signaling pathway For DC activation and maturation experiments (Figure 2A and B), bone marrow DCs (1 × 106) from C57BL6 mice were stimulated with 50 ng/mL LPS. Simultaneously, DCs were treated with annexin A5 protein at concentrations of 1, 5 and 10 µg/mL. All groups were incubated at 37°C for 18 h. (A) DC activation levels: 18 h after the treatment, concentrations of proinflammatory cytokines IL6, TNFα, and ILIO were assessed in supernatants using ELISA. Each cytokine levels are illustrated using bar graphs. Data represent the mean ± S.E.M. of three independent experiments. LPS treatment was used as a positive control and immature DC culture supernatant served as a negative control. * — indicates significant differences (P < 0.05) from data obtained during the LPS only challenge. (B) DC maturation levels: 18 h after the treatment, maturation surface markers CD40, CD80 and CD86 on DCs were measured using flow cytometry. The bar graph illustrates the mean fluorescence intensity (MFI) of each surface marker. LPS treatment was used as a positive control and immature DCs served as a negative control. * — Indicates significant differences (P < 0.05) from data obtained during the LPS-only challenge. (C) TLR4 signaling pathway protein levels: Bone marrow DCs (4 × 106) were stimulated with 50 ng/mL LPS. Concomitantly, the annexin A5 protein was added at a concentration of 20 µg/mL and incubated at 37°C for indicated times (10, 30 and 60 min). Cell lysates were collected after indicated time points and various TLR4 signaling proteins, namely MAPKs (ERK, p38 JNK), IkB-α and phosphorylated MAPKs (p-ERK, p-p38 and p-JNK), were detected by Western blot, β-actin was used as a loading control. Similar results were obtained in three separate experiments.