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. Author manuscript; available in PMC: 2023 Aug 18.
Published in final edited form as: Cell. 2022 Jul 30;185(17):3214–3231.e23. doi: 10.1016/j.cell.2022.06.038

Figure 5. N-GSDMD directly mediates depolarization of macrophage mitochondrial membranes following AIM2 activation.

Figure 5.

A. GSDMD association (anti-GSDMD; PE, x-axis) with the mitochondrial network measured by flow cytometry of isolated mitochondria 4h after AIM2 activation +1 μM disulfiram or DMSO (vehicle) B. TMRE staining of WT BMDMs treated with 1 μM disulfiram or DMSO (vehicle), followed by AIM2 activation for 4h C. Schematic of recombinant proteins used in in vitro experiments D. GSDMD cleavage in vitro via recombinant wt or mutCASP11 in the presence of mitochondrial extracts from WT and Lrrk2G2019S BMDMs (n=2) E. FACS plot of TMRE staining of mitochondria isolated from WT BMDMs in the presence of full length GSDMD and wtCASP11 or mutCASP11. 1h incubation. Quantitation at lower right. F. As in E. but with mitochondria isolated from WT and Lrrk2G2019S BMDMs. 30 min incubation G. N-GSDMD mitochondrial association in WT and Lrrk2G2019S BMDMs via biochemical fractionation and immunoblot during AIM2 activation +/− 25 μM menadione. VDAC1; control for mitochondrial membrane enrichment. (right) N-GSDMD relative to VDAC1 normalized to WCL H. As in A but 2h after AIM2 activation, WT BMDMs +/− 25 μM menadione Statistical analysis: n=3 or more unless otherwise noted. Statistical significance determined via a one-way ANOVA with Sidak’s post-test (A, B, E, F, H).