Figure 2.

Insulin significantly reduces the NLRP3-mediated formation of the ASC pyroptosome. (A) Immunoblotting for caspase-1 (p20 and caspase-1 precursor), IL-1β (p17 and IL-1β precursor), and NLRP3 of the supernatant (upper panels) and cell lysates (lower panels) from THP-1 monocytes pretreated with insulin (0, 1, 5, and 15 IU/ml), followed by stimulation with LPS (1 μg/ml) for 6 h and ATP (5 mM) for 30 min. (B) Quantification of immunoblotting was determined by densitometric analysis, and each result was normalized to the levels of the vehicle control group. (C) Immunofluorescence microscopy of LPS (1 μg/ml for 24 h) and ATP (5 mM for 30 min)-stimulated THP-1 monocytes pretreated with insulin (0, 5, and 15 IU/ml) for 30 min, and subsequently immunostained for ASC (green) and DNA (DAPI, blue). Scale bars, 10 μm. White arrows indicate ASC specks. (D) The quantification represents the percentages of cells with an ASC speck, with ≥100 cells counted from 10 random fields in each experiment. (E) ASC oligomerization and redistribution assay in THP-1 cells treated as in (A). Immunoblotting analysis of ASC in cross-linked pellets (upper panels) and in cell lysates (lower panels). The quantification data are presented as mean values (± SDs) derived from experiments performed three independent times under identical conditions; Immunoblotting results as shown in (A, E) are one representative of three independent experiments; **p < 0.01 (one way ANOVA with Dunnett’s multiple comparisons test).