FIGURE 6.

Poly(I:C) pretreatment primes B-cells to the activation by ssDNA via up-regulation of UNC93B1. A–C, Ramos-Blue cells were stimulated with poly(I:C) (20 μg/ml), ODN2216 (20 μg/ml), ODN10104 (20 μg/ml), and R-848 (20 μg/ml) for the indicated times. A, NF-κB/AP-1-dependent secreted embryonic alkaline phosphatase (SEAP) activity (A₆₃₀) was measured in supernatants. UNC93B1 (B) and TLR9 (C) mRNA were subsequently measured by real-time PCR. D and E, cells were stimulated with poly(I:C) (20 μg/ml) + DMSO alone or pretreated with bafilomycin A (Baf.) for the indicated times. UNC93B1 (D) and IFN-β (E) mRNA were measured by real-time PCR. The results are represented by mean values with S.D. from triplicate wells. The representative data from three experiments are shown. F–I, Ramos-Blue B-cells were pretreated with medium (Ø) or first agonist for 0–12 h and subsequently treated with second agonist for additional 12 h. Agonist used were poly(I:C) (0.5 μg/ml) and ODN2216 (0.5 μg/ml). F, the first agonist was poly(I:C), and the second agonist was ODN2216. G, the first agonist was poly(I:C), and the second agonist was also poly(I:C). H, the first agonist was ODN2216, and the second agonist was also ODN2216. I, the first agonist was ODN2216, and the second agonist was poly(I:C). Results shown in F–I were all preformed in one experiment setting. The results are represented by mean values with S.D. from triplicate wells. The representative data from three experiments are shown.