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. 2018 Jan 18;2018:6346734. doi: 10.1155/2018/6346734

Figure 3.

Figure 3

Alkylation-mediated NLRP3 inflammasome inhibition of ALE. (a) Mature IL-1β and active caspase-1 in culture supernatants of LPS-primed BMDMs treated with ALE or forskolin in the presence or absence of H89 for 30 min, followed by nigericin (Nig) for 1 h, were immunoblotted. (b–g) LPS-primed BMDMs were treated with ALE, Bay11-7082 (Bay), G5, or KCl in the presence or absence of L-cysteine (L-cys) (b and d) or glutathione (GSH) (c and e) for 15 min, followed by nigericin for 1 h, and culture supernatants and crosslinked lysates were analyzed by immunoblotting. (f) ATPase activity of NLRP3 in the presence or absence of ALE was determined by luminescence by using the ADP-Glo assay. The data represent the mean ± SD of two independent experiments. ∗p < 0.05 and ∗∗p < 0.01 compared with vehicle treated control (g) BMDMs were treated with ALE for 30 min before stimulation with LPS for the indicated time, and phospho-IκB-α and IκB-α in cell lysates were analyzed by immunoblotting. (h and i) The LPS-primed BMDMs were stimulated nigericin in the presence or absence of ALE. Levels of mitochondrial ROS in BMDM were analyzed by MitoSOX labeling. (h) The representative flow cytometric histogram and (i) quantitative analysis of mean fluorescence intensities of two independent experiments are shown.