Extended Data Fig. 1: Ion flux is not required for TLR9-induced inflammasome.
From: Eukaryotic ADCY7 catalyzes the production of c-di-AMP to activate the NLRP3 inflammasome
(a) Tlr9+/+ and Tlr9–/– BMDM cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Cell viability was determined by an ATP quantification assay in cell pellets. (b) Tlr9–/– BMDM cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Otherwise, cells were stimulated with 1 μM control ODN or CpG-ODN and 1 μg/ml LPS for 6 h, followed by culture media replacement with PBS containing 10 μM nigericin for 30 min. Cell viability was determined by an ATP quantification assay in cell pellets. (c) Tlr9+/+ and Tlr9–/– BMDM cells were stimulated with 1 μM control ODN or CpG-ODN for 6 h. Intracellular potassium levels were determined through an inductively coupled plasma optical emission spectrometry and shown as relative levels. d–f, Wild-type BMDM cells primed with 1 μg/ml LPS for 3 h were stimulated with 10 μM nigericin for 30 min. 50 mM KCl was added to the medium 30 min before the addition of nigericin. Otherwise, cells were co-stimulated with 1 μM CpG-ODN and 50 mM KCl for 6 h. (d) Cell culture supernatants were collected for an ELISA assay to determine the secreted IL-1β protein levels. (e) Cytotoxicity was determined by LDH release assay in cell culture supernatants. (f) Cell viability was determined by an ATP quantification assay in cell pellets. g, h, Wild-type BMDM cells primed with 1 μg/ml LPS for 3 h were stimulated with 2 mM ATP for 30 min. 50 mM KCl was added to the medium 30 min before the addition of ATP. Otherwise, cells were co-stimulated with 1 μM CpG-ODN and 50 mM KCl for 6 h. (g) Cytotoxicity was determined by LDH release assay in cell culture supernatants. (h) Cell viability was determined by an ATP quantification assay in cell pellets. In a–h, data were shown as means ± SD (n = 4, biological replicates). Two-tailed Student’s t-test were used.
