Fig. 4.

Qu inhibits the LPS-induced NLRP3 inflammasome and related pyroptosis in BV2 cells. Qu (30 and 60 μM, 1 h)-pretreated BV2 cells were stimulated with LPS (100 ng/ml, 24 h) and ATP (5 mM, 30 min). (a–c) Expression of NLRP3, pro-caspase-1, pro-IL-1β, full-length GSDMD and cleaved GSDMD N-terminal in cell lysate (Ly) was detected by an immunoblot assay (a). The relative expression levels were quantified (c). Actin was used as an internal loading control. The cleaved caspase-1 and cleaved IL-1β in the supernatant (SN) were also detected by an immunoblot assay (a) and quantified as normalized to the control group (b). (d) ELISA of IL-1β in supernatants from different-treated BV2 cells. (e–f) Treated BV2 cells were costained with YO-PRO-1 (green), a small (629.3 Da) membrane impermeable but pyroptosis-pole permeable dye, and Eth-D2 (red), a larger (1292.7 Da) membrane and pyroptosis-pole impermeable dye, to identify cells with discrete membrane pores. Hoechst staining (blue) indicates total cells. Treatment with 0.1% Triton was used as the positive control. Scale bar, 40 μm. (f) Quantitative analysis of cells that stained positive for YO-PRO-1 and negative for Eth-D2. A total of 15 fields/group (3 wells/group and 5 fields/well) cells were counted. Data are shown as the mean ± SEM of three to four independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 by one-way ANOVA followed by Tukey's post hoc test. Ctl, untreated control; Qu, quercetin; LPS, lipopolysaccharide; Ly, lysate; SN, supernatant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)