Figure 2.

Nigericin- and cSiO₂-induced IL-1β release are LPS- and ASC-dependent in RAW MΦs. (A,C) RAW-ASC cells were pretreated with 20 ng/ml LPS for 2 h, incubated with 0, 2.5, or 5.0 μM nigericin for 45 min (A), or 0, 10, or 25 μg/ml cSiO₂ for 4 h (C), and then release of IL-1β measured. (B,D) RAW-ASC and RAW-WT were pretreated with VEH or LPS for 2 h, incubated with VEH or 10 μM nigericin (B) or 25 μg/ml cSiO₂ (D). IL-1β release was assessed at the indicated times. (E) Pro-IL-1β was present in the cell extracts (CE) of RAW-ASC MΦs treated with LPS, but only secreted into the supernatant (SN) with nigericin or cSiO₂ treatment. IL-1β in the supernatant contained both the precursor and cleaved forms. (F) Bone marrow-derived macrophages were pretreated with VEH or LPS (20 ng/ml) for 2 h, incubated with VEH or 5 μM nigericin or 25 μg/ml cSiO₂ and IL-1β release then assessed at 45 min or 4 h, respectively. Data presented as mean ± SEM, n = 3. ND = not detectable. Asterisks indicate significant differences between cell type (B,D) or treatment group (A,C,E) (*p < 0.05, **p < 0.01). Different letters indicate significant differences between treatment groups within each cell type (B,D) (p < 0.05). ELISA data are representative of three independent experiments. Western blots are representative of two independent experiments.