Extended Data Fig. 8: ADCY7 deficiency hampers NLRP3 inflammasome.
From: Eukaryotic ADCY7 catalyzes the production of c-di-AMP to activate the NLRP3 inflammasome
(a) Adcy7–/– BMDM cells were rescued with lentiviruses carrying the indicated ADCY7 isoforms for 3 days. Cells were then stimulated with 1 μM CpG-ODN for 6 h and immunostained with antibodies against TLR9 and ADCY7. Nuclei were stained with DAPI. (b) Adcy7–/– BMDM cells rescued with wild-type or enzymatic-mutant ADCY7 were stimulated with 1 μM CpG-ODN for 6 h. Cells were lysed and immunoprecipitated with a control IgG or antibody against TLR9. Precipitates were immunoblotted as indicated. (c) Adcy7–/– BMDM cells rescued with the indicated ADCY7 truncations were stimulated with 1 μM CpG-ODN for 6 h. Cells were lysed and immunoprecipitated with a control IgG or antibody against TLR9. Precipitates were immunoblotted as indicated. (d) Wild-type BMDM cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Otherwise, cells were transfected with 10 μg/ml cGAMP using DOTAP for 6 h. Cells were lysed and run on a Native-PAGE for STING dimerization. Cells lysates were also immunoblotted as indicated. (e) Wild-type, Adcy7–/– and Tlr9–/– mice (n = 4 for each group) were intraperitoneally injected with D-GalN (20 mg per mice) and CpG-ODN (20 nmol per mice). Meanwhile, mice were injected with 100 nmol c-di-AMP through hydrodynamic tail vein injection. Serum levels of IL-1β were determined 6 h post injection. In a, scale bar, 10 µm. In e, data were shown as means ± SD (n = 4, biological replicates). Two-tailed Student’s t-test were used.
