Figure 3

The effect of BAG on TNF-α production is TLR9 dependent. (A) RAW264.7 cells were pretreated with TLR9 inhibitors: 0, 1, 10, or 100 nM ODN2088 or 0, 0.1, 1, or 10 μM CQ for 1 h, followed by stimulation with 10 μg/ml BAG for an additional 24 h. (B) RAW264.7 cells were transfected with TLR9 siRNA or control siRNA for 48 h. Then, the cells were stimulated with 1 μM CpG ODN2395 as a positive control or 10 μg BAG for an additional 24 h. (C) BMDM from TLR9 WT and KO mice were stimulated with 0, 0.1, 1, or 10 μg/ml BAG for 24 h. (D) BMDM from TLR9 WT and KO mice were stimulated with 1 μM CpG ODN2395 or 0.1 μg/ml PAM3CSK4 for 24 h. (E) BMDM from TLR9 WT and KO mice were infected with increasing numbers of B. anthracis spores (MOI of 1, 3, or 10) for 8 h. At the end of the incubation period, culture supernatants were collected and subjected to ELISA. *P <0.05 and **P <0.01 as compared with the relevant control group.