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. 2021 Oct 21;12:752482. doi: 10.3389/fimmu.2021.752482

Figure 1.

Figure 1

Higher doses of nigericin induce IL-1β release in the absence of specks. (A) THP-1 cells were treated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 72 h and treated with 5 μM colchicine for 1 h followed by stimulation with different doses of nigericin for 2 h Culture supernatant was used for IL-1β ELISA. (B) THP-1 cells were treated as described in (A). Following nigericin stimulation, cells were fixed and stained for NLRP3 and ASC and analyzed for specks by microscopy. A minimum of 600 cells were analyzed per condition and counted for the presence of speck manually. Data represented as mean ± SEM for two independent experiments. N.D., not detected. (C) Representatives micrographs for data shown in (B). Cells were stained for NLRP3 (red), ASC (green), and DAPI (blue). Scale bar = 25 nm. Single channel staining shown in Figure S3 . (D) THP-1 cells were primed with 100 nM PMA for 72 h and treated with 5 μM colchicine for 1 h followed by stimulation with nigericin at the indicated concentrations for 2 h. Cells were stained for ASC (red) and DAPI (blue). Scale bar = 25 nm. Representative micrographs are shown. (E) THP-1 cells were primed with 100 nM PMA for 72 h and treated with 5 μM colchicine for 1 h followed by stimulation with nigericin at the indicated concentrations for 2 h. Culture supernatant was used for IL-1β ELISA. (F) THP-1 cells were treated with 100 ng/ml of LPS for 3–4 h and treated with 5 μM colchicine for 1 h followed by stimulation with nigericin at the indicated concentrations for 2 h. Culture supernatant was used for IL-1β ELISA. (G) THP-1 cells were stimulated with PMA as in (E) and lysed with 0.09% Triton X-100. IL-1β lysates were diluted 1:5 in assay buffer and measured by ELISA. Data represented as mean ± SEM for a minimum of three independent experiments, unless otherwise mentioned. **p < 0.01, ***p < 0.001, ****p < 0.0001 for comparison with respective untreated controls; two-way ANOVA followed by Dunnett’s (A) multiple comparison tests. # p < 0.05, ### p < 0.001 Student’s t-test for comparison between colchicine-treated cells stimulated with or without 5 μM nigericin.