Figure 5.

Baicalin blocked adenosine triphosphate (ATP)-induced ASC speck formation and oligomerization. Bone marrow-derived macrophages were primed with LPS (500 ng/ml) for 4 h, pretreated with baicalin (0.6 mM) for 1 h, and triggered with ATP (3 mM) for 30 min in the absence of LPS. (A) Representative immunofluorescence images showing ASC (red) and NOD-like receptor (NLR) family, pyrin containing domain 3 (NLRP3) (green) subcellular distributions. Nuclei (blue) were stained with Hoechst 33342. Merged images were also presented to show the co-localization of ASC specks and NOD-like receptor (NLR) family, pyrin containing domain 3 (NLRP3) dots in the cytoplasm. Yellow arrows indicate the ASC specks, and the enlarged insets show cells with an ASC speck. Scale bars, 20 µm. (B) Quantification of cells with ASC specks relative to the total number of cells in five randomly chosen fields with each containing ~50 cells. (C) Western blot analysis for ASC in Triton-X 100 insoluble pellets cross-linked with disuccinimidyl suberate. The experiments were performed three times independently, with one representative experiment shown. Data are shown as mean ± SD (n = 3). ***P < 0.001.