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. 2023 Nov 9;14:7246. doi: 10.1038/s41467-023-43180-8

Fig. 5. Mitochondrial pore formation, GsdmD-dependent depolarization, GsmdD cleavage and mitochondrial association of N-GsdmD in NLRP3-activated cells.

Fig. 5

Examples of mitochondria with discontinuities in the outer membrane (OM) in cryo-ET reconstructions of in ASC-mCerulean iBMDMs stimulated with LPS and nigericin. Dashed boxes denote the OM discontinuities. Scale bars, 100 nm. See Supplementary Fig. 8 for additional examples of OM discontinuities. Z-stack series and cryo-ET density measurements for the OM gap boxed in green in (a). Scale bar, 50 nm. The graph shows the cryo-ET density at the OM gap site (yellow) and at an adjacent site with intact inner and outer membranes (pink). Cryo-ET density is expressed as the density in the intermembrane space (IMS) divided by the density in a nearby cytosolic area. The areas in which IMS density was measured are circled in the Z-stack panels (yellow, gap site; pink, intact site). See Source Data File for source data. Flow cytometry histograms of WT iBMDMs, (c), or GsdmD-/- iBMDMs, (d), stained with tetramethylrhodamine (TMRM), a mitochondrial membrane potential reporter. See Supplementary Fig. 10 for additional controls. Vertical axes indicate cell count. e Immunoblots of subcellular fractionation of WT iBMDMs 60 min after stimulation with LPS and nigericin. The GsdmD N-terminal domain (N-GsdmD) is enriched in the mitochondrial fraction. Shown below are immunoblots for Tom20 (a mitochondrial protein), GAPDH (a cytosolic protein), and CD14 (a plasma membrane protein). See also Supplementary Fig. 9. The blots are representative of four independent experiments with similar results.