Fig 2: Nigericin leads to NLRP3 localisation at centrosomal and non-centrosomal locations.THP1GFP-NLRP3 cells were left untreated or treated with ZVAD (50 µM, 40 min) or MCC950 (10 µM, 15 min) prior to treatment with nigericin (10 µM, 45 min) to activate the NLRP3 inflammasome (A–D). Images show NLRP3 (green), ASC (Red) and PCNT (purple). Nuclei are shown in blue. ASC speck positive cells were quantified and plotted as percentages versus total number of cells (B). Percentages of cells with NLRP3 at centrosome relative to cells with centrosome and cells with no PCNT in total cells were calculated respectively (C, D). THP1GFP-NLRP3 cells were left untreated or treated with MCC950 (10 µM, 15 min) before stimulation with nigericin (10 µM) at different time points as indicated (E, F). Percentages of cells with ASC specks, or with NLRP3 and PCNT, or with no PCNT in total cells were calculated. 300 cells were counted and analysed per experiment, N = 3, Biologically independent experiments.

Fig 3: Detection of ASC, caspase-1 and IL-1β in liver biopsies at the protein level.a Representative western blot for the detection of caspase-1 (pro- and active forms) and ASC in liver biopsy extracts from 4 different donated livers collected before donation (T1) or after cold ischemic storage (T2). Β-actin was used as a loading control. Full-length western blots are published as a Supplementary File. b Concentration of IL-1β in the 4 extracted tissues as detected by ELISA. c Correlation matrix among the expression of different NLRP3 inflammasome-related genes detected in liver tissue collected before donation (T1) or after cold ischemic storage (T2).

Fig 4: Hepatocyte inflammasome activation detection during donation as biomarker for liver transplant outcome.Kaplan-Meier survival curve for the presence of high (> median) or low (< median) amounts of ASC specks (a) or IL1B gene expression (b) in donated fixed liver tissue collected before donation (T1) or after cold ischemic storage (T2).

Fig 5: Nigericin treatment of THP-1 cells results in loss of centrosomal integrity.THP1ATCC cells were left untreated or treated with nigericin (10 µM) at the indicated time points to activate the NLRP3 inflammasome, then cell lysates and supernatants were analysed for PCNT, γ-tubulin, and β-actin protein expression by western blot (A–F, N = 3). Lysates and supernatants were analysed for PCNT and γ-tubulin as well as loading control β-actin (42 kDa) (D). Whole well lysate with centrifugation step (E) and whole cell lysate with no centrifugation step (F) were also analysed for those proteins. Cell death was measured by LDH assay and shown as percentage relative to total cell death (A). Caspase-1 activity in supernatants was measured and shown as fold change relative to control (B). IL-18 in the supernatants was measured by ELISA (C). THP1ATCC cells were stimulated with nigericin (10 µM) for 45 min or 90 min. G–K Immunofluorescence was used to analyse the centrosomal proteins including PCNT (G), γ-tubulin (H) and ninein (I) as well as ASC to determine the NLRP3 inflammasome activation. Percentages of ASC speck, PCNT, γ-tubulin or ninein positive cells relative to total cells (J) and PCNT, γ-tubulin or ninein positive cells in ASC positive cells (K) were quantified. N = 3 biologically independent experiments. For multiple comparisons, one‐way ANOVA with the Dunnett’s test for time response in THP1ATCC cells was applied. Data was shown as mean ± S.D., *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001 were considered statistically significant when compared to vehicle treatment (time 0).