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. Author manuscript; available in PMC: 2020 Nov 4.
Published in final edited form as: Nat Immunol. 2020 May 4;21(7):736–745. doi: 10.1038/s41590-020-0669-6

Figure 5.

Figure 5

GSDMD pore formation is the main target of disulfiram. (a-e) LPS-primed THP-1 cells, pretreated or not with 30 μM disulfiram or z-VAD-fmk for 1 hr and stimulated with nigericin or medium, were analyzed for ASC specks (a, b), caspase-1, GSDMD and pro-IL-1β cleavage and IL-1 release by immunoblot of whole cell lysate (WCL) or culture supernatants (Sup) (c), and redistribution of GSDMD to the plasma membrane (d, e). (a) shows representative images of ASC specks (arrowheads) and (b) shows mean ± s.d. percent of cells with ASC specks analyzed 20 min after adding nigericin. The ratios of cells with ASC specks were calculated by counting 30 high-power fields for each sample in 5 independent experiments and analyzed using two-tailed Student’s t-test. In (c) WCL and Sup, harvested 1 hr after adding nigericin, were immunoblotted with the indicated antibodies. The GSDMD antibody used was generated in house (Extended Data Fig. 6). In (d, e) cells were fixed 30 min after adding nigericin and stained for GSDMD using a previously unreported monoclonal antibody generated in house (Extended Data Fig. 6). Shown are representative confocal microscopy images (d) and quantification (e) of the proportion of cells with GSDMD membrane staining and pyroptotic bubbles. Arrows indicate GSDMD staining of pyroptotic bubbles. The ratios of cells with GSDMD membrane staining were calculated by counting 40 high-power fields for each sample in 5 independent experiments and analyzed using two-tailed Student’s t-test. (f) Mouse iBMDMs were pretreated with disulfiram, Bay 11–7082, necrosulfonamide (NSA) or z-VAD-fmk for 1 hr before adding Nigericin. Whole cell lysates and culture supernatants, harvested 1 hr after adding nigericin, were immunoblotted with the indicated antibodies. Graphs show the mean ± s.d; data are representative of three independent experiments. Data in (c, f) are representative of three independent experiments. *P < 0.05, **P < 0.01. (g) Model of inflammasome pathway steps and their inhibition by disulfiram, with a main effect on GSDMD.