Figure 3.

Metformin treatment increased adenosine triphosphate (ATP)-induced ASC speck formation and interleukin (IL)-1β secretion in bone marrow-derived macrophages (BMDMs). (A) Mouse BMDMs were seeded in glass-bottomed dishes and primed with lipopolysaccharide (500 ng/ml, in complete DMEM medium) for 4 h, and then treated with metformin (2 mM, in Opti-MEM) for 1 h. Subsequently, 2 mM ATP was added to the medium for 30 min. The distribution of ASC (red) was revealed by immunofluorescence microscopy with the nuclei being stained with Hoechst 33342 (blue). The images for ASC and nuclei were captured, respectively, and merged together. The experiment was performed twice, with one representative set of images shown. The yellow arrows indicate ASC specks. An enlarged inset for each image with an ASC speck is also shown. Scale bar, 20 µm; Met, metformin. (B) The ratios of cells containing ASC specks were calculated by the number of cells with ASC specks relative to the total number of cells from five random fields each containing about 50 cells. Data are shown as mean ± SD (n = 5). (C) Quantification of soluble IL-1β levels in the cell culture supernatants by cytometric bead array assay. Data are shown as mean ± SD (n = 3). ***P < 0.001.