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. 2013 Apr 3;288(19):13534–13550. doi: 10.1074/jbc.M113.457218

FIGURE 7.

FIGURE 7.

Calcium signaling orchestrates DAI-mediated macrophage activation induced by ALD-DNA. A and B, HEK293 cells were infected with 0.1 μg of pNF-κB-Luc (A) or pIRF3-Luc (B), plus 0.1 μg of pDAI for 36 h. The cells were pretreated with EGTA (1 mm), BAPTA-AM (50 μm), CsA (3 μg/ml), or CGP37157 (10 μm) for 2 h, then left stimulated with UnALD-DNA (1 μg/ml) or ALD-DNA (1 μg/ml) for another 12 h. Luciferase activities were measured and normalized to Renilla luciferase activities. C and D, the siControl or siDAI-expressing macrophages were infected with 0.1 μg of pNF-κB-Luc (C) or pIRF3-Luc (D) for 36 h. The cells were pretreated with EGTA (1 mm), BAPTA-AM (50 μm), CsA (3 μg/ml), or CGP37157 (10 μm) for 2 h, then stimulated with ALD-DNA (1 μg/ml) for another 12 h. Luciferase activities were measured and normalized to Renilla luciferase activities. E, the siControl or siDAI-expressing macrophages were pretreated with EGTA (1 mm), BAPTA-AM (50 μm), CsA (3 μg/ml), or CGP37157 (10 μm) for 2 h, then stimulated with ALD-DNA (1 μg/ml) for another 24 h. ELISA analysis was performed to detect the levels of TNF-α, IL-6, and IFN-β. F and G, the siControl or siDAI-expressing macrophages were infected with 0.1 μg of pNF-κB-Luc (F) or pIRF3-Luc (G) for 36 h. The cells were pretreated with valinomycin (1 nm) or thapsigargin (20 nm) for 2 h, then stimulated with ALD-DNA (1 μg/ml) for another 12 h. Luciferase activities were measured and normalized to Renilla luciferase activities. H, the siControl or siDAI-expressing macrophages were pretreated with valinomycin (1 nm) or thapsigargin (20 nm) for 2 h, then stimulated with ALD-DNA (1 μg/ml) for another 24 h. ELISA analysis was performed to detect the levels of TNF-α, IL-6, and IFN-β. Data are mean ± S.E. of three independent experiments. **, p < 0.01; ***, p < 0.001; NS, not significant. I, the siControl or siDAI-expressing macrophages were stimulated with ALD-DNA (1 μg/ml) for 30 min. The levels of intracellular calcium were measured. J, the siControl or siDAI-expressing macrophages were stimulated with ALD-DNA (1 μg/ml) for 30 min. Western blot analysis was performed to determine the levels of CaMKII-α phosphorylation (T286). K, the RAW264.7 cells were pretreated with BAPTA-AM (50 μm) for 2 h, then left stimulated with ALD-DNA (1 μg/ml) for another 24 h. Western blot analysis was performed to determine the levels of RIP1. Data are representative of results obtained in three independent experiments.