Figure 5
Compound effects on NLRP3 downstream assays (caspase-1, IL1-β, pyroptosis). ASC-mCherry iBMDM were treated with the indicated compounds (10 µM) at the same time as LPS (1 µg/ml, 2 h) and subsequently stimulated with nigericin (10 µM, 2 h). (a) Immunoblot of pro-IL-1β and cleaved IL-1β (IL-1β p17), pro-caspase-1 (p45) and caspase-1 (p20) in the cell lysates and supernatants after cell stimulation with LPS + nigericin in the presence or absence of compounds. β-actin was used as a loading control for the cell lysate samples. The IL-1β and caspase-1 bands are cropped from their respective blot images and the full-length blots are presented in Figure S6. (b) Caspase-1 activity using Caspase-1 Glo assay with the clear bars are (−) Ac-YVAD-CHO (caspase-1 inhibitor), while dashed lines bar are (+) Ac-YVAD-CHO, (c) IL-1β release using ELISA and (d) LDH release. In all experiments (b–d) data is presented as the mean ± SEM, n = 4 independent experiments and one-way ANOVA with post-hoc Dunnett’s test was utilized to calculate significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data in (b–d) was analyzed using GraphPad Prism version 7 software (https://www.graphpad.com/scientific-software/prism/).
