Fig 2: Oxidative phosphorylation profile. Laser desorption/ionization-time of flight (MALDI-TOF/TOF) mass spectrometry, RT-qPCR and western blot analysis for studying the oxidative phosphorylation pathway in muscle tissues of normal weight (N) and overweight (OW) patients. (A) Levels of proteins implied in pyruvate dehydrogenase activity regulation (pyruvate dehydrogenase (PDH), phospho-pyruvate dehydrogenase (p-PDH) and its ratio (p-PDH/PDH)). Bar charts show means of semi-quantitative optical density (O.D.) ± SEM. Representative immunoblots of PDH and p-PDH. Ponceau staining was used as a loading control (B) The amount mitochondrial content is marked by the protein levels of TOM20. Bar charts show the means of semi-quantitative optical density (O.D.) ± SEM. (C) RT-qPCR was used to confirm mitochondrial DNA (mtDNA) content. mtDNA content was compared against genomic DNA (nDNA). Data are expressed as the mean ± SEM (D) Levels of subunits from the protein complexes of the mitochondrial electron transport chain (NADH dehydrogenase (ubiquitone) 1b subcomplex 8 (NDUFB8) from complex I (CI), iron sulfur subunit (SDHB) from complex II (CII), ubiquinolcytochrome c reductase core protein II (UQCRC2) subunit from complex III (CIII), cytochrome c oxidase subunit I (MTCO1) from complex IV (CIV), and ATP synthase subunit α (ATP5A) from complex V (CV), isoform c of the ATP synthase subunit α from complex V and the H+ transporting F1 complex subunit β from complex V). Bar charts show the means of semi-quantitative optical density (O.D.) ± SEM. Representative immunoblots of subunits from the protein complexes of the mitochondrial electron transport chain (CI, CII, CIII, CIV and CV). Ponceau staining was used as a loading control and TOM20 as a mitochondrial marker **, P < 0.01; ***, P < 0.001.

Fig 3: Carboxyl-terminal helices of Dictyostelium discoideum MCU (DdMCU) confer EMRE-independent Ca2+ uptake to Homo sapiens MCU (HsMCU).(A) Schematic shows helices and sheets of HsMCU and DdMCU as predicted by PSIPRED. Two transmembrane domains are labeled. (B, C) Schematic summarizes the domain structure of HsMCU, DdMCU, and the chimeric proteins. FLAG-tagged proteins were stably expressed in MCU KO and EMRE KO HEK293T cells. Mitochondrial Ca2+ uptake rates in control WT and chimera expressing cells were measured and normalized to those of WT cells (n = 3–4). Expression of chimeras was detected by Western blotting using anti-FLAG antibody. TOM20 serves as loading control. Data information: in (B, C), data are presented as mean ± SD.

Fig 4: Tom20-induced translocation of Bax to mitochondria contributes to pyroptosis. Melanoma A375 cells were treated with CCCP (20 μM), FeSO4 (100 μM), or CCCP/FeSO4 for 6 h to detect the translocation of Bax to mitochondria, cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. To detect the effects of the Tom20 point mutants Tom20C13S and Tom20C21S, Tom20 was knocked down in A375 cells, and Tom20WT or its point mutants Tom20C13S and Tom20C21S were separately transfected into cells. a Bax translocated to mitochondria upon CCCP/FeSO4 stimulation as shown by confocal microscopy. b Knockdown of Tom20 blocked the translocation of Bax to mitochondria upon CCCP/FeSO4 treatment. The mitochondrial fraction in cells was prepared. c Knockdown of Bax blocked the CCCP/FeSO4-induced mitochondrial aggregation. d-g After knocking down Bax, the CCCP/FeSO4-induced cytochrome c release detected in the cytosol fraction was diminished (d), the cleavage of caspase-3 and -9 was attenuated (e), the cell viability was rescued (f), the cell morphologies were reversed from pyroptosis to normal state, and LDH release and GSDME cleavage were also abolished (g). h Effects of the mutants Tom20C13S and Tom20C21S on the translocation of Bax to mitochondria in response to CCCP/FeSO4 stimulation. Hsp60 was used to detect the mitochondria, and DAPI was used to display the nuclei by confocal microscopy. Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. ***P < 0.001

Fig 5: ROS induces Tom20 oxidation in response to iron stimulation. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) for 2 h, followed by CCCP (20 μM), FeSO4 (100 μM), or CCCP/FeSO4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. To detect the effects of the Tom20 point mutants Tom20C13S, Tom20C21S and Tom20C100S, Tom20 was knocked down in A375 cells, and Tom20WT or its point mutants Tom20C13S, Tom20C21S and Tom20C100S were separately transfected into cells. a FeSO4 induced Tom20 oxidation, which was attenuated by NAC even in the presence of CCCP. Western blotting was performed under reducing and non-reducing conditions to determine the Tom20 oxidation status. b Mutation of either Cys13 or Cys21 in Tom20 abolished the CCCP/FeSO4-induced Tom20 oxidation. c CCCP/FeSO4 could not induce Tom20C13S and Tom20C21S accumulation. d Mutation of either Cys13 or Cys21 in Tom20 blocked mitochondrial aggregation as shown by confocal microscopy. Hsp60 represents the mitochondria, and DAPI was used to display the nuclei. e-h Mutation of either Cys13 or Cys21 in Tom20 abrogated the CCCP/FeSO4-induced cytochrome c release as detected in the cytosol fraction (e), cleavage of caspase-3 and -9 (f), LDH release and cell death (g), and reversed the cell morphology from pyroptosis to normal state (h). Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. ***P < 0.001