Fig. 5: Gasdermins and MLKL disrupt mitochondria.

A, B Wild type BMDMs were treated with vehicle or 300 nM 5ZOZ for 5 h. The cytosolic including plasma membrane (C) and the mitochondrial (M) proteins were resolved by SDS-PAGE with and without a reducing agent (2-mercaptoehtanol), and analyzed by Western blotting. Oligomerized bands of GSDMD and MLKL and N-terminal GSDME are indicated. Mitochondrial marker TOM20 and cytosolic marker GAPDH are shown as loading controls. B 20 µM pan-caspase inhibitor, emricasan, was also treated for 5 h. Smaller amounts of proteins were loaded to avoid anti-P-MLKL background bands. C HeLa-RIPK3 cells were transfected with vectors expressing GFP alone or GFP-GSDMD. At 24 h post-transfection, cells were treated with vehicle or 50 ng/ml TNF and 1 μM 5ZOZ (TZ) for 5 h. Cytosol and mitochondrial fractions were analyzed in reducing and non-reducing conditions by Western blotting. TOM20 and GAPDH are shown as loading controls for mitochondria and cytosol, respectively. The estimated molecular weights of GFP-GSDMD and cleaved GFP-GSDMD-N are about 75 and 50 kDa, respectively. D HeLa-RIPK3 with GFP-GSDMD were treated with vehicle (top and 2nd panels) or with 50 ng/ml TNF and 1 µM 5ZOZ (3rd and bottom panels) for 5 h. Immunofluorescence staining of anti-TOM20 (red) and GFP-GSDMD (green) was analyzed by confocal microscopy. 2nd and bottom panels show enlarged images of top and 3rd panels, respectively. Scale bars, 10 µm. Control GFP expressing cells are shown in Supplementary Fig. S5A. Volumetric (3D) image of the TNF and 5ZOZ treated cell are shown in Supplementary Video 1. E Wild type BMDMs were treated with vehicle or 300 nM 5ZOZ for 4 h. Mitochondrial respiration was determined by Seahorse assay (Oligo, oligomycin; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rote/Ant, rotenone + antimycin A). A representative analysis is shown (upper panel). Oxygen consumption rate (OCR) was normalized with protein concentration per well (lower graph). Bars represent mean values of basal respiration and maximal respiration from three independent Seahorse assays. Students’ t-test, ***, p < 0.001; **, p < 0.01; *, p < 0.05; NS not significant.