Extended Data Fig. 6: ADCY7 catalyzes c-di-AMP production.
From: Eukaryotic ADCY7 catalyzes the production of c-di-AMP to activate the NLRP3 inflammasome
(a) c-di-AMP was docked into the mouse ADCY7 or mouse ADCY4 structure (Alphafold: AF-Q91WF3-F1-v4) and vacuum electrostatics was shown. (b) Adcy7–/– BMDM cells were rescued with wild-type or enzymatic-mutant ADCY7 without transmembrane domains. Cells were stimulated with 1 μM control ODN or 1 μg/ml E. coli genomic DNA for 6 h. Cells were lysed and the intracellular c-di-AMP levels were determined by an ELISA assay. (c) Human macrophages were infected with lentiviruses carrying Cas9 and an sgRNA against human ADCY7. ADCY7+/+ and ADCY7–/– human macrophages were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Cells were lysed and the intracellular c-di-AMP levels were determined by an ELISA assay using an antibody specific for c-di-AMP. (d) ADCY7+/+ and ADCY7–/– human macrophages were stimulated with 1 μM control ODN or 1 μg/ml E. coli genomic DNA for 6 h. Cells were lysed and the intracellular c-di-AMP levels were determined by an ELISA assay using an antibody specific for c-di-AMP. (e) ADCY7–/– human macrophages were rescued with wild-type or enzymatic-mutant ADCY7 without transmembrane domains. Cells were stimulated with 1 μM control ODN or 1 μg/ml E. coli genomic DNA for 6 h. Cells were lysed and the intracellular c-di-AMP levels were determined by an ELISA assay using an antibody specific for c-di-AMP. (f) Wild-type BMDM cells primed with 1 μg/ml LPS for 3 h were stimulated with 10 μM nigericin for 30 min. Otherwise, cells were stimulated with 1 μM CpG-ODN for 6 h. Cells were lysed and the intracellular c-di-AMP levels were determined by an ELISA assay using an antibody specific for c-di-AMP. (g) Wild-type human macrophages primed with 1 μg/ml LPS for 3 h were stimulated with 10 μM nigericin for 30 min. Otherwise, cells were stimulated with 1 μM CpG-ODN for 6 h. Cells were lysed and the intracellular c-di-AMP levels were determined by an ELISA assay using an antibody specific for c-di-AMP. In b–g, data were shown as means ± SD (n = 4, biological replicates). Two-tailed Student’s t-test were used.
