Fig 2: BAT characteristics of deep cervical human depot. A) Histological H&E staining and UCP1 immunostaining of tissue sections obtained from human superficial and deep cervical fat depots in multi and unilocular patients. Scale bars are 100 µm. B) Representative Western blot of unilocular (Uni) and multilocular (Multi) deep and superficial cervical lysates and quantification of mitochondrial protein levels in the Multi samples (n = 4). C) Mitochondrial content in superficial and multilocular deep cervical fat layer in Multi patients (n = 6). D) Mitochondrial respiration in superficial and multilocular deep cervical frozen lysates in Multi patients (n = 5). E) Representative transmission electron microscopy of cervical deep fat depot from a multi patient. Scale bar = 1000 nm. F) Mitochondrial (GRP75, red), lipid droplet surface (PLIN1, green) immunostaining, and nuclear staining (DAPI, blue) in the indicated samples coming from a multi patient. Scale bars are 10 µm; Scale bar in zoomed insert is 1 µm *, p < 0.05 paired Student's t-test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig 3: RIFS validation in pathophysiological relevant mouse and human disease models A–DComplex I-, II-, and IV-dependent respiration by protein (A), MTDR mitochondrial content (B), Complex I-, II-, and IV-dependent respiration corrected by mitochondrial mass (C), and Western blot followed by quantification (D) in heart homogenates from WT and Ndufs4 KO mice.EComplex I-, II-, and IV-dependent respiration by protein in Polg mutant (Polg MT) and control heart homogenates.FMTDR mitochondrial content in Polg MT and control heart homogenates.GComplex I-, II-, and IV-dependent respiration corrected by mitochondrial mass.HWestern blot followed by quantification in heart homogenates from WT (control) and Polg MT mice.IQuantification of Complex I-, II-, and IV-dependent respiration in zebrafish muscle homogenate from young and old fish.JQuantification of Complex I- and IV-dependent respiration using RIFS in zebrafish deyolked embryo homogenate acutely treated with rotenone before freezing.KComplex I to Complex IV ratio in zebrafish deyolked embryo homogenate in the indicated conditions.LComplex I activity measure spectrophotometrically in zebrafish deyolked embryo homogenate in the indicated conditions.MRepresentative H&E sections from human donors show the presence of multilocular adipose tissue in the pheochromocytoma (Pheo) sample. UCP1-DAB staining (central panels) and Grp75/mortalin fluorescence staining (bottom panels) from control and Pheo patients.NWestern blot followed by quantification in homogenates from control and Pheo patients.OComplex I-, II-, and IV-dependent respiration by protein in periadrenal adipose tissue homogenates from control and Pheo human patients.PMitochondrial content by MTDR quantification in homogenates from control and Pheo human patients.QQuantification of complex activities (I, II, and IV) in MAS versus MAS supplemented with Cyt c and ALA buffer in isolated and cryopreserved human monocytes, lymphocytes, and platelets.Data information: Biological replicates: (A–I), n = 3; (J–L), n = 3 (each n represents a pool of 300 embryos); (N–P), n = 8; and (Q), n = 3–6 per group. Every biological replicate represents the average of at least four technical replicates. Data are the mean ± SEM. Western blots illustrate a representative blot of three independent experiments. The stainings are representative of eight control and four Pheo cases. Three representative fields were imaged and assessed for every biological sample. Contrast and brightness settings are the same across the samples. Scale bars represent 100 µm. For calculating statistics, the number of replicates used is the biological replicates indicated above. We used the following tests: two-way ANOVA with Sidak's multiple comparison test in panels (A–E, G, and O); one-way ANOVA Tukey's multiple comparison test in panels (J–L); unpaired t-test in panels (F, H, N, and P). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available online for this figure.

Fig 4: Absence of SFXN1 leads to complex-III-related defects(A) Steady-state abundance of select mitochondrial proteins, including OXPHOS components, subunits of import machineries, and other proteins in the IMM and matrix.(B) Immunoblotting for Fe-S containing subunits of respiratory complexes in mitochondrial extracts. GRP75 served as loading control.(C) Immunoblotting for mtDNA-encoded subunits in mitochondrial extracts. GRP75 served as loading control.(D) Densitometric analysis of bands for select proteins in (A), (B), and (C). Protein levels in WT were set to 1.0 (mean ± SEM, n = 4).(E) Electron flow through the respiratory chain in intact mitochondria. Base buffer contains 10 mM pyruvate, 2 mM malate, and 4 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Injections of complex inhibitors/substrates were performed as indicated. rot, rotenone; succ, succinate; AA, antimycin A; asc/TMPD, ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine.(F) Spectrophotometric assays using detergent-solubilized mitochondria to monitor individual complex activities. CI, oxidation of NADH to NAD+; CII, ubiquinol production; CIII, cytochrome c reduction; CIV, cytochrome c oxidation (mean ± SEM, n = 4).(G) 1D BN assembly. Mitochondria solubilized in 1% (w/v) digitonin were resolved on a 4%–16% BN gel and immunoblotted for the indicated subunits.(H) Mitochondria solubilized in 1% (w/v) digitonin were resolved by 2D BN/SDS-PAGE and immunoblotted for the indicated subunits. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; unpaired Student’s t test.

Fig 5: BAT characteristics of peri-adrenal fat in Pheo patients. A) Histological H&E staining and lipid size quantification of tissue sections obtained from human subcutaneous (SC) and peri-adrenal (pADR) control and Pheo patients (n ≥ 5). Scale bars are 100 μm. B) UCP1 immunostaining and quantification of subcutaneous (SC) and retroperitoneal peri-adrenal (pADR) tissue sections from control and Pheo patients (control, n = 9; Pheo, n = 7). Scale bars = 100 μm. C) Representative Western blot and quantification of UCP1 levels in SC and pADR tissue lysates from control and Pheo patients (control, n = 13; Pheo, n = 8). D) Mitochondrial content in tissue lysates from SC and pADR samples from control and Pheo patients (control, n = 15; Pheo, n = 9). E) Representative Western blot and quantification of mitochondrial respiratory protein levels in SC and pADR tissue lysates from control and Pheo patients (control, n = 12; Pheo, n = 7). F) Representative images of whole mount-periadrenal adipose tissue from control subjects and Pheo patients stained with DAPI (blue), BODIPY493/503 (green), and primary Ab against GRP75 (red). Contrast in the red channel (GRP75) was adjusted to help visualize mitochondrial localization in control samples. Scale bars are 10 μm. G) Mitochondrial respiration in SC and pADR frozen lysates from control and Pheo patients (control, n = 15; Pheo, n = 10). **, p < 0.01; ****, p < 0,0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)