Figure 4.

Au4.5 activates the NLRP3 inflammasome through its inhibitory effect on macroautophagy. (A) Immunoblot analysis of cell lysates from BMDCs primed with LPS (100 ng/mL, 3 h) and stimulated by Au4.5 (200 μg/mL) for 6 h, followed by immunoprecipitation with anti-NLRP3 and immunoblot analysis (antibodies, left margin). (B) Immunoblot of 293CIA cells transfected with plasmid for HA-NLRP3 and treated with Au4.5 or nigericin for 6 h. Cell lysates were immunoprecipitated with anti-LC3 and immunoblot for NLRP3. (C) Confocal microscopy analysis of colocalization of LC3 and NLRP3 in 293CIA cells transfected with plasmids encoding EGFP-LC3 and NLRP3, followed by Au4.5 stimulation for 6 h. Cells were fixed and stained with DAPI (blue) and anti-NLRP3 followed with RPE-conjugated anti-mouse IgG antibody (red). Scale bar, 10 μm. Arrows indicate colocalization. (D,E) ELISA analysis (D) and immunoblot (E) of BMDCs primed with LPS, followed by Au4.5, ATP, or nigericin stimulation in normal, starvation condition, or along with autophagy inducer 4-OH tamoxifen treatment (5–10 μM, 12 h prior to stimulation and also applied during stimulation). Immunoblot of pro-Caspase-1 and Caspase-1 p10 were performed. Niger represents nigericin (E). (F,G) 293CIA, 293CIA^LC3−/−, 293CIA^{ATG16L−/−}, and 293CIA^BECN1−/− cells were transfected with Flag-NLRP3 and then stimulated by Au4.5 or nigericin for 6 h. Relative quantity of Caspase-1 p20/22 bands are shown on the right side (F). Induction of IL-1β in cell supernatants was analyzed by ELISA (G). Data are representative of three experiments (mean ± SD). *p < 0.05, **p < 0.01, ***p < 0.001.