459200

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of SDHA in isolated mitochondria using an SDHA Monoclonal antibody (Product # 459200). Lane 1: Isolated mitochondria from Human heart at 5 µg, Lane 2: Isolated mitochondria from Bovine heart at 4 µg, Lane 3: Isolated mitochondria from Rat heart at 10 µg, Lane 4: Isolated mitochondria from Mouse heart at 10 µg, Lane 5: Isolated mitochondria from HepG2 at 20 µg.

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Experimental details: Western blot analysis of SDHA in isolated mitochondria using an SDHA Monoclonal antibody (Product # 459200). Lane 1: Isolated mitochondria from Human heart at 5 µg, Lane 2: Isolated mitochondria from Bovine heart at 4 µg, Lane 3: Isolated mitochondria from Rat heart at 10 µg, Lane 4: Isolated mitochondria from Mouse heart at 10 µg, Lane 5: Isolated mitochondria from HepG2 at 20 µg.

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Experimental details: Knockdown of SDHA was achieved by transfecting SH-SY5Y with SDHA specific siRNAs (Silencer® select Product # S12650, S12651). Western blot analysis (Fig. a) was performed using Membrane enriched extracts from the SDHA knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with SDHA Monoclonal Antibody (2E3GC12FB2AE2) (Product # 459200, 0.1µg/mL ) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to SDHA.

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Experimental details: Western blot was performed using Anti-SDHA Monoclonal Antibody (2E3GC12FB2AE2)(Product # 459200) and a 70 kDa band corresponding to SDHA was observed across cell lines and tissue tested. Membrane enriched extracts (30 µg lysate) of SH-SY5Y (Lane 1), HeLa (Lane 2), LnCAP (Lane 3), SK-OV-3 (Lane 4), Hep G2 (Lane 5) and A549 (Lane 6); and tissue extract of Mouse Kidney (Lane 7) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (0.1 µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

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Experimental details: Immunofluorescent analysis of SDHA in human embryonic lung-derived fibroblasts using an SDHA Monoclonal antibody (Product # 459200) at a concentration of 0.2 µg/mL. Cultured human embryonic lung-derived fibroblasts (strain MRC5) were fixed, permeabilized and then labeled with an SDHA Monoclonal antibody (Product # 459200) at a concentration of 0.2 µg/mL, followed by an AlexaFluor® 488-conjugated-goat-anti-mouse IgG2a isotype specific secondary antibody at a concentration of 2 µg/mL.

Supportive validation

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Experimental details: Immunofluorescent analysis of SDHA in human embryonic lung-derived fibroblasts using an SDHA Monoclonal antibody (Product # 459200) at a concentration of 0.2 µg/mL. Cultured human embryonic lung-derived fibroblasts (strain MRC5) were fixed, permeabilized and then labeled with an SDHA Monoclonal antibody (Product # 459200) at a concentration of 0.2 µg/mL, followed by an AlexaFluor® 488-conjugated-goat-anti-mouse IgG2a isotype specific secondary antibody at a concentration of 2 µg/mL.

Supportive validation

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Experimental details: Immunohistochemical analysis of SDHA in skeletal muscle using an SDHA Monoclonal antibody (Product # 459200). Fixed frozen skeletal muscle tissue sections are from a patient with a single large deletion of the mtDNA. All muscle fibers exhibit complex II immunoreactivity, consistent with the nuclear DNA-encoded expression pattern of this and all other subunits of complex II.

Supportive validation

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Experimental details: Immunofluorescent analysis of SDHA in human embryonic lung-derived fibroblasts using an SDHA Monoclonal antibody (Product # 459200) at a concentration of 0.2 µg/mL. Cultured human embryonic lung-derived fibroblasts (strain MRC5) were fixed, permeabilized and then labeled with an SDHA Monoclonal antibody (Product # 459200) at a concentration of 0.2 µg/mL, followed by an AlexaFluor® 488-conjugated-goat-anti-mouse IgG2a isotype specific secondary antibody at a concentration of 2 µg/mL.

Supportive validation

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Experimental details: Fig 1 Characterization of TFAM and Twinkle (TW) mice. (A) Expression of human TFAM (hTFAM) in left ventricle (LV) and aorta in TFAM and wild-type (WT) control mice. (B) mtDNA copy number in myocardium from TFAM and TW mice by real-time PCR (n = 4). (C) Expression of endogenous murine TFAM (mTFAM) and mitochondrial complex proteins in LV of TFAM and TW mice. (D) Transcription of mtDNA-encoded genes in TFAM and TW mice (n = 6). Data are expressed as mean +- SEM. * P < 0.05 vs. WT, ** P < 0.01 vs. WT, analyzed by Student's t- test.

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Experimental details: Figure 5. Liver-specific Cluh deletion affects mitochondrial distribution and structure, assembled respiratory supercomplexes, and respiratory capacity. (A) Representative confocal images of livers of 8-wk-old mice of the indicated genotypes. To analyze mitochondrial morphology, mice were crossed with a stop-mito-YFP reporter line activated by Cre recombination. n = 4. Bars, 5 um. (B) Oxygen consumption of mitochondria isolated from livers of 8-wk-old mice. State III respiration was measured in the presence of pyruvate, malate, glutamate, and ADP (complex I [CI]), followed by addition of succinate (complex I + complex II [CI + CII]). The proton leak was measured after addition of oligomycin, whereas maximal respiration was assessed by CCCP titration. n = 5. Graph shows means +- SEM. **, P

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Experimental details: Figure 7. Cluh -deficient MEFs mimic liver phenotypes. (A) Oxygen consumption of intact MEFs cultured in glucose or galactose medium. The proton leak was measured after the addition of oligomycin, whereas maximal respiration was assessed by CCCP titration. n >= 4. (B) Mitochondrial morphology in MEFs transfected with mito-mCherry. Graphs show the mean aspect ratio (area/perimeter) on the x axis and the number of mitochondria on the y axis for individual cells from three independent experiments. Right panels show representative images of mitochondrial morphology at the indicated time points. Bar, 12 um. (C) Growth curves of MEFs cultured in glucose or galactose medium during five consecutive days. n = 3. (D) Representative images of LD staining in MEFs grown in glucose medium. Nuclei were stained with DAPI (blue), and LDs were stained with BODIPY 493/503 (green). Bar, 20 um. (E) Quantification of LD staining shown in D. 50 cells were analyzed per genotype per experiment. Graph shows the mean area of LDs per cell. n = 3. (A, C, and E) Error bars are means +- SEM. *, P

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Experimental details: Figure 3 MCU Delta NTD overexpression has a dominant-negative effect on mitochondrial Ca 2+ uptake A Co-immunoprecipitation of MCU WT -Flag or MCU Delta NTD -Flag with MCU WT - GFP . HeLa cells were transiently co-transfected with MCU WT - GFP and MCU WT -Flag/ MCU Delta NTD -Flag. MCU Delta NTD migrated farther than MCU WT , indicating an apparent molecular weight difference of 9 kD a in SDS - PAGE (see also Fig EV4 A). MCU WT - GFP with MCU WT -Flag or MCU Delta NTD -Flag was precipitated from the cell lysates with an anti- GFP antibody. The precipitates were separated on SDS - PAGE and immunoblotted with the antibodies indicated. B Co-immunoprecipitation of MCU Delta NTD -Flag with MCU Delta NTD - GFP . HeLa cells were transiently co-transfected with MCU Delta NTD -Flag and MCU Delta NTD - GFP . MCU Delta NTD - GFP with MCU Delta NTD -Flag was precipitated from cell lysates with an anti- GFP antibody. The precipitates were separated on SDS - PAGE and immunoblotted with the indicated antibodies. C HeLa cells were transiently transfected with MCU WT -Flag or MCU Delta NTD -Flag. After isolation and solubilization of crude mitochondria, the lysates were subjected to BN-PAGE and immunoblotted with anti-Flag and anti- MCU antibodies to detect ectopic MCU WT -Flag, MCU Delta NTD -Flag and endogenous MCU . MCU WT and MCU Delta NTD were detected at apparent molecular weights of 480 and 440 kD a, respectively. The shift shown in MCU Delta NTD complex correlated with the difference

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Experimental details: Fig. 1 Focal depletion of mitochondria in skeletal muscles of a human R350P desminopathy patient and R349P desmin knock-in mice. a Cytochrome C oxidase (COX, brown ) and succinate dehydrogenase (SDH, blue ) double-stains of transverse and longitudinal cryosections from a German patient harboring the heterozygous R350P (c.1049G>C) desmin missense mutation [ 3 ]. Note the multiple rubbed-out areas ( asterisks ) devoid of COX and SDH enzyme activities demonstrating the absence of mitochondria. The right image represents a false color representation of two superimposed serial cryosections stained for COX ( magenta ) and desmin ( black ). Sarcoplasmic desmin-positive protein aggregates clearly display a distribution which is independent from the mitochondrial lesion pathology. b , c COX ( b ) and SDH ( c ) stains of transverse and longitudinal cryosections from 3-month-old R349P (c.1045_1047delAGG>insCCC) desmin knock-in mice. Fibers of homozygote (HOM) animals display large and small areas of diminished enzyme stains ( asterisks ). Furthermore, muscle fibers in homozygote animals show an abnormal, thread-like distribution of mitochondria. Heterozygous (HET) mice showed no overt pathology as compared to wild-type (WT) littermates

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Experimental details: Figure 2 The mitochondrial translation machinery regulates global mitochondrial activity and Emu -myc cell growth A. Emu- myc lymphoma cells were transduced with shRNAs against Ptcd3 , Mrps5 , Mrps27 , Renilla luciferase (Ren) or a control empty vector (EV). The number of viable cells at each time point (top) was determined by trypan blue staining. For each single shRNA the efficiency of knockdown was evaluated by real-time qPCR three days post-transduction (bottom). Transcript abundance is expressed as mean +- s.d. of triplicate measurements, expressed relative to the EV control and normalized to Rplp0 . B. Doubling time was calculated from the growth curves in A., using the formula described in experimental procedures. C. Cell death was evaluated by Annexin V and propidium iodide (PI) staining at the 48hrs time-point. The graph shows the percentage of Annexin V+/PI- (black) and Annexin V+/PI+ cells (grey) corresponding to early and late apoptotic cells, respectively. D. Western blot analysis of components of the Electron Transport Chain (ETC) Complexes I-IV following 48hrs of knock-down. As loading controls, we used antibodies against cytoplasmic Vinculin and the mitochondrial Voltage-dependent anion channel (VDAC). E. Real-time qPCR quantification of mtDNA, normalized to nuclear DNA. Results are shown as mean +- s.d. from triplicate measurements. F. Oxygen consumption rate (OCR) was determined as described in experimental procedures at 48hrs. Data were normalized to total

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Experimental details: Figure 3 Tigecycline impairs the growth of Emu -myc lymphoma cells and inhibits translation of mitochondrial proteins A. Dose-response curve of Emu- myc lymphoma cells treated with the indicated doses of Tigecycline (Tig), Linezolid (Lin) or Cloramphenicol (Cam) for 48hrs. Data are shown as cell numbers (left panel) or viability (right panel), as percentage of untreated cells (mock-treated with DMSO). Cell number and viability was determined by trypan blue staining. B. Oxygen consumption rate (OCR) measured as defined in Figure 2F on Emu- myc lymphoma cells treated with the indicated antibiotics or carrier (DMSO). To measure spare respiratory capacity, cells were treated with the ETC uncoupler 2,4-Dinitrophenol (DNP). C. Western blot analysis of components of the ETC complexes I-IV. Cells were treated with the indicated concentration of Tigecycline for 24 or 48hrs. The loading controls are Vinculin and VDAC, as in Figure 2D . D. Mitochondrial membrane potential was determined by staining with the cationic cyanine dye DilC1(5). E. Cleaved caspase 3/7 activity was determined with the caspase 3/7 glo assay luminescent kit. Measurements in D and E were taken after 6 hrs of treatment. Means, s.d. and statistical significance are as defined in Figure 2 . See also Supplementary Figures S3 and S4 .

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Experimental details: Figure 1 Exercise training increase mitochondrial oxidative phosphorylation complexes in an AMPK alpha2-dependent manner . Protein levels of oxidative phosphorylation complexes, (A) Complex I, (B) Complex II, (C) Complex III, (D) Complex IV, (E) Complex V (oligomycin-sensitivity conferring protein subunit, OSCP), and (F) Cytochrome C were evaluated in quadriceps muscle of sedentary (control) or exercise trained female WT and AMPK alpha2 KD mice ( n = 13-15). Values are mean +- SEM. * indicates vs. WT control ( p < 0.05) analyzed by unpaired t -tests.

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Experimental details: Figure 4 Repeated treatment with AICAR increases abundance of mitochondrial electron transport chain proteins in an AMPK alpha2-dependent manner . WT and AMPK alpha2 KD male mice were given daily subcutaneous injections with AICAR (500 mg/kg body weight) or saline for 4 weeks. (A-E) Protein abundance of oxidative phosphorylation Complexes I-V and (F) Cytochrome C was measured in quadriceps muscle ( n = 7-8). A significant interaction effect (treatment x genotype; p < 0.05) was observed for Cytochrome C. Values are mean +- SEM. * indicates vs. WT saline ( p < 0.05) analyzed by t -tests, ** indicates vs. WT saline ( p < 0.01), and ++ indicates genotype effect within AICAR treated animals ( p < 0.01).

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Experimental details: Figure 3 csPIMT -/- hearts show significant mitochondrial damage. ( A ) Quantification of mRNA levels of Atp1a2, Atp2a1, Ryr2 and Tfam genes. Each group was analyzed using 5 different mice (assayed individually) and the values are expressed as the mean +- SD. * p < 0.05, ** p < 001, NS: not significant; ( B ) Western blot showing protein levels for PCS (palmitoyl-CoA synthetase; 62 Kda), complex II30 and 70, MH/ECHS1 (mitochondrial enoyl-CoA hydratase; 31 Kda), MTP (mitochondrial trifunctional protein; 100 Kda) and CPT1alpha (carnitine palmitoyltransferase; 88 Kda)). The protein extracts were prepared from 5 hearts pooled together. The protein expression of each gene was normalized to GAPDH. Percent decrease as compared WT controls were as follows: PCS, 67%; complex II30 and 70, 28% and 38%; MTP, 26%, and CPT, 42%; ( C , D ) display the electron micrographs of 6-month csPIMT fl/fl and csPIMT -/- mouse hearts. Red arrows in D indicate abnormal sarcomeres and H zone absent. Yellow arrows point to lipid droplets and damage in mitochondria.

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Experimental details: Figure 1. IKKalpha regulates mitochondrial biogenesis and fiber type specification in vivo. (A) Hind limbs from four independent litters of IKKalpha +/+ and IKKalpha -/- embryos (total n = 8) were isolated at E20. Cytochrome c and COX Va were measured by real-time quantitative RT-PCR. (B) Additional tissues from E20 limbs from A were homogenized and probed by Western blotting. (C) Total DNA was extracted from E20 limbs, and real-time RT-PCR analysis was performed to quantitate mitochondrial and nuclear (GAPDH) genome copy number. Results are means of five mice per genotype and calculated as fold mitochondrial over genomic DNA. P = 0.004. (D) AAV-GFP and AAV-IKKalpha viruses were injected into the TA. Mice were sacrificed after 3-4 mo, and protein lysates were prepared and analyzed by Western blotting. Shown is a representative blot from five independent experiments. (E and F) After similar viral injections as described in D, mitochondria were fractionated, and protein concentrations or DNA content was determined and normalized to total protein/DNA content (E; n = 5 per group; P < 0.005) or TA muscles were analyzed by EM (F). Bar, 1 um. Parallel muscles were stained for SDH. Bar, 200 um. Arrowheads point to individual mitochondria. (G) TA muscles were isolated from AAV-GFP- and AAV-IKKalpha-injected mice ( n = 3 per group, repeated five times), and myosin isoforms were measured by real-time RT-PCR. The data shown are from a single representative experiment out of three replica

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Experimental details: Fig. 4 Citrate synthase and complex I activities and expression levels of single subunit proteins of respiratory complexes I-V in R349P desmin knock-in mice. a Determination of the citrate synthase activity by a spectrophotometric assay using identical amounts of total protein extracts. Note the marked activity reduction in homozygous mice. For this approach, soleus muscles obtained from five mice of each genotype (WT, HET, HOM) were pooled and subjected to four-time repeated measurements. Column chart shows mean values of these technical replicates. b Determination of complex I activity by a colorimetric assay normalized to muscle weight. Though this analysis, performed in duplicate on non-pooled samples derived from three animals per genotype, showed a reduction of complex I activity in homozygous mice, the data failed to reach statistical significance. An additional normalization of the obtained complex I values to the citrate synthase values resulted in similar activity levels for the three genotypes (data not shown). Column chart shows mean values and standard errors. c Immunoblotting using antibodies directed against specific complex I-V proteins revealed increased, decreased, and missing signals of complex I subunit proteins in heterozygous and homozygous mice, whereas the signal intensities of complex II-V subunit proteins showed no obvious differences. Desmin immunoblotting confirmed a markedly decreased level of mutant desmin in homozygous mice. In heterozygous mice

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Experimental details: Figure 5 Substitution of Ser92 to Ala in MCU NTD reduced mitochondrial Ca 2+ uptake activity A Expression of MCU WT -Flag, MCU S 92A -Flag and MCU K 180A -Flag in stable MCU - KD HeLa cells. The solubilized mitochondrial fraction from each sample was subjected to BN - PAGE and immunoblotted with anti-Flag antibody. The lysates of each sample were subjected to SDS - PAGE and detected with the indicated antibodies. Flavoprotein subunit of complex I is used as a loading control of each mitochondrial fraction. B Interaction of MCU mutants with MCU WT was not altered. After co-expression of MCU WT and MCU mutants, co-immunoprecipitation was performed. The precipitates were separated by SDS - PAGE and immunoblotted with the indicated antibodies. C, D Mitochondrial Ca 2+ uptakes evoked by 100 muM histamine were quantified by measuring the peak amplitudes of the traces. F 0 is initial fluorescence intensity. F and F max indicate fluorescence intensity at each time point and maximal fluorescence intensity after the stimulation, respectively. (F max -F 0 )/F 0 indicates the maximal Ca 2+ concentration evoked by the stimulation (mean +- SEM , n = 13-15, * P < 0.05 versus control sh RNA -expressing cells. # P < 0.05 versus stable MCU - KD cells. SS P < 0.05 versus MCU WT -rescued stable MCU - KD cells). Unpaired two-sided Student's t -test was used to calculate statistical significance.

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Experimental details: Figure 3 SDHA and SDHB immunoreactivity. ( A , C , E )--SDHA immunohistochemistry. ( B , D , F )--SDHB immunoreactivity. ( A ) and ( B ) represent a case with both SDHA and SDHB immunoreactivity; ( C ) and ( D ) have SDHA but lack SDHB immunoreactivity; ( E ) and ( F ) represent a case lacking both SDHA and SDHB immunoreactivity. SDHA--Succinate dehydrogenase A, SDHB--Succinate dehydrogenase B, Original objective 40x.

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Experimental details: Figure 3 Expression of the human parkin variant parkinQ311X induces early mitochondrial dysfunction in SNc DA neurons. ( a ) Representative Western blots showing the levels of mitochondrial proteins (OPA1, SDHA, VDAC) in lysates prepared from the substantia nigra of WT or parkinQ311X mice at 1 month of age. Samples were run in triplicate, with each lane loaded with lysate from an individual mouse. The histograms on the right show the mean +-SEM calculated from densitometer quantification (** p < 0.01 unpaired Student's t -test). ( b ) Representative TEM images showing mitochondria in SNc DA neurons of WT (control, i,ii ) and parkinQ311X ( iii,iv,v ) mice. The mitochondria in control specimens show the typical rounded/tubular morphology ( i,ii ). The mitochondria in parkinQ311X mice display marked ultrastructure disruption ( iii,iv,v ) and a lack of the typical crystal organization found in the control tissue: 26% of the mitochondria in parkinQ311X DA neurons displayed an almost complete loss of outer membrane shape and form ( iii ). Other mitochondria in the parkinQ311X DA neurons retained outer membrane integrity but demonstrated ultrastructural damage: dilation of intracrystal spaces, loss of matrix density, and deposits of electron-dense material within the matrix (vesicular mitochondria) ( iv ). Swollen, clear mitochondria showed herniation of the swollen matrix covered by the inner membrane through the ruptured regions of the outer membrane ( v ). The histograms on the r

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Experimental details: FIGURE 8. FAHD1 immunofluorescence. Confocal images of immunofluorescence staining of HUVEC for FAHD1 ( green , left column ), three different localization markers ( red , center column ), and the resulting merge ( right column ) are shown. Complex II and complex IV were used as mitochondrial markers, and catalase was used as peroxisomal marker.

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Experimental details: Figure 5 Changes in VDAC, complex II and V proteins, and mtDNA expression following 3 h OGD. Representative western blots for VDAC, Complex II, and Complex V with their corresponding beta-actin blots (A, B, C), together with relative intensity values for these proteins (D). VDAC and Complex V proteins showed increased expression, whereas Complex II protein expression did not change significantly following OGD. (E) Shows the changes in mtDNA expression following 3 h of OGD. MtDNA/nuclear DNA ratio significantly increased 12 h following 3 h OGD. *p

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Experimental details: Figure 2 CI*CIV-SC content diminishes in the brain of Ndusf4 KO mice (A) OCR measurement in ex vivo hippocampal brain sections from control (WT) and Ndufs4 knockout mice (KO) (WT, n = 3; Ndufs4 KO, n = 3, two-way RM ANOVA). In the violin plot, numbers represent p values. (B) Immunoblot analysis of samples from WT and KO mice using antibodies against ETC subunits (CI, NDUFB8; CII, SDHA; CIII, UQCRC2; CIV, MTCO1; CV, ATP5A; WT, n = 2; Ndufs4 KO, n = 2). GAPDH was used as loading control. Densitometry is relative to WT samples and reported as mean +- SEM (nd, not determined). (C) BN-PAGE and composite image of immunoblots obtained using antibodies against NDUFB8 (CI) and MTCO1 (CIV, cropped bands). Samples were from WT and KO brain homogenates (WT, n = 3; Ndufs4 KO, n = 3). (D and E) Coimmunostaining and fluorescence profiles of (D) VDAC1 and NDUFB8 and (E) TOM20 and MTCO1 in control mouse brain sections (two-way ANOVA, *p < 0.05). Scale bar, 5 mum. (F) Colocalization between mitochondria (TOM20) and CI*CIV-SCs (PLA). Scale bar, 1 mum. (G and H) Representative pictures of hippocampal CA1 pyramidal neurons from (G) WT and (H) KO mice stained with DAPI (nucleus), NeuN (neurons), TOM20 (mitochondria), and PLA (CI*CIV-SCs). Scale bar, 5 mum. (I-N) Quantification of (I) area of the soma, (J) mitochondrial area, (K) mitochondria/soma ratio, (L) PLA dots normalized to the soma, (M) PLA quantification of CI*CIV-SCs normalized to the mitochondrial area, and (N) linear correlation of PLA

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Experimental details: Figure 4 CI*CIV-SC remodeling occurs during iPSC differentiation (A) OCR measurement of iPSCs and iPSC-derived smNPCs using a conventional Seahorse protocol (n = 3). (B) Mitochondrial spare respiratory capacity in human iPSCs and smNPCs. Percentage is relative to their respective basal OCRs (Student's t test, ****p < 0.0001). (C) Basal OCR in iPSCs and smNPCs (Student's t test, ****p < 0.0001). (D) Immunoblots using antibodies against OXPHOS system components in iPSCs and smNPCs. Densitometry is relative to iPSCs and reported as mean +- SEM (n = 3, Student's t test, ***p < 0.001, *p < 0.05). (E) BN-PAGE and corresponding immunoblots using antibodies against MTCO1 and NDUFB8. (F) Representative pictures of iPSCs and smNPCs stained with DAPI (nucleus, blue), TOM20 (mitochondria, green), and PLA (CI*CIV-SCs, red). Oct3 and nestin staining (both in gray) was used as markers of pluripotency and differentiation, respectively. Scale bar, 5 mum. (G) Quantification of PLA dots normalized to mitochondrial area in iPSCs and smNPCs (n = 3; iPSCs = 20 cells; smNPCs = 17 cells; Student's t test, ***p < 0.001). (H) OCR measurement of WT (P) and two independent clones of NDUFS4 KO iPSCs (KO 1 and KO 2 ). (I) Basal OCR and mitochondrial spare capacity of WT and NDUFS4 KO iPSCs (fold change, relative to WT; two-way RM ANOVA, ***p < 0.001, *p < 0.05; ns, not significant). (J) Immunoblot analysis of homogenates from parental and NDUFS4 KO iPSCs using antibodies against ETC subunits (CI, NDUFS4,

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Experimental details: Figure 5. [4Fe-4S] cluster proteins are severely affected in heart Sod2 knockout mice indicating strong increase in superoxide levels. ( A ) Aconitase activity from purified mitochondria from control (pp) and Sod2 loxP x Ckmm cre mice (pp, cre). White bar indicates activity in control samples ( n = 6, 9-10 week old) and gray bar in Sod2 loxP x Ckmm cre samples ( n = 6, 9-12 week old). Activity is normalized to control. ( B ) Western blot analysis of ACO 2 (aconitase) protein levels from purified mitochondria of control (pp) and Sod2 loxP x Ckmm cre mice (pp, cre) (9-10 week old). ATP5A and Coomassie-stained membrane were used as loading controls. ( C ) Oxygen consumption rate of isolated heart mitochondria from control (pp, white bars, n = 9, 9-11 week old) and Sod2 loxP x Ckmm cre mice (pp, cre, gray bars, n = 9, 9-12 week old). Isolated mitochondria were incubated with complex I (PMG) or complex II (SUCC) substrates. Each set of substrates was successively combined with ADP (to assess the phosphorylating respiration, PMG3, SUCC3), oligomycin (to assess the non-phosphorylating respiration PMG4, SUCC4) and CCCP (to assess uncoupled respiration PMGc, SUCCc). ( D ) Activity of the respiratory chain complexes I (CI), II (CII), IV (CIV) and the activity from complex II to III (CII-III) of heart mitochondria from control (pp, write bars, n = 3, 11-week old) and Sod2 loxP x Ckmm cre mice (pp, cre, gray bars n = 3, 11-12 week old). Citrate synthase

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Experimental details: Fig. 6. Proteasome inhibition decreases mitochondrial respiration and impairs mitochondrial respiratory complex protein homeostasis in NRK cells. A : digitonin-permeabilized NRK cells (5 x 10 6 ) were treated with vehicle control (DMSO) or the ChT-L proteasome inhibitor bortezomib (BTZ; 10 nM) for 24 h. High-resolution respirometry was used to determine oxygen flux through complexes I, II, and III. Values are expressed as % of control of 4 independent experiments. Differences between groups were compared with an unpaired Student's t- test. *Means are significantly different ( P < 0.05) compared with control. B : ATPase activity of ATP synthase (complex V) was assayed in NRK cells treated with DMSO (vehicle control) or BTZ for 24 h. Renal cell extracts (10 mug) with or without oligomycin (20 muM) were incubated with the enzyme mix in the presence of ATP and NADPH (MitoCheck Complex V Activity Assay kit, Cayman). NADH oxidation rate was monitored at 340 nm for up to 30 min with a BioTek Gen5 microplate reader. Bar graph shows oligomycin-inhibitable ATPase activity expressed as % of control ( n = 3). Differences between groups were compared with a Student's t -test. *Means are significantly different ( P < 0.001). C and D : Western blots of SDHA and ATP5B in soluble ( C ) and insoluble fractions ( D ) of NRK cell extracts after treatment with vehicle control (Con; DMSO) or BTZ (10 nM) for 24 h. Representative blots of 4 independent experiments are shown

Supportive validation

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Experimental details: Figure 2 Expression of selected genes from pyrimidine nucleotide synthesis pathways in human neuroblastoma SH-SY5Y cells. ( A , B ) Representative optic microscopy images of ( A ) undifferentiated and ( B ) neuron-differentiated SH-SY5Y cells. White arrows point to neurites. ( C , D ) Representative immunofluorescence microscopy images of anti-TUBB3 stained ( C ) undifferentiated and ( D ) neuron-differentiated SH-SY5Y cells. Inset: enlarged figure detail. ( E ) Representative image of a flow cytometry histogram of anti-TUBB3 stained undifferentiated (white) and neuron-differentiated (green) cells. ( F , G ) Representative immunofluorescence microscopy images of anti-TH stained ( F ) undifferentiated and ( G ) neuron-differentiated SH-SY5Y cells. Inset: enlarged figure detail. ( H ) Representative image of a flow cytometry histogram of anti-TH stained undifferentiated (white) and neuron-differentiated (red) cells. ( I ) Graph showing the change of fluorescence intensity (FI) in TUBB3 and TH levels after neuronal differentiation. Dashed line (100 %) represents TUBB3 or TH mean values of undifferentiated cells. Bars indicate mean values and standard deviations in differentiated cells. N = 11. *: p < 0.05 (versus undifferentiated cells). ( J ) Oxygen consumption of (U) undifferentiated and ( D ) neuron-differentiated cells. N = 3. *: p < 0.05 (versus undifferentiated cells). ( K ) DHODH and UCK2 mRNA levels in (U) undifferentiated and ( D ) neuron-differentiated cells. Points re

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4. Depletion of mitochondrially encoded components of CIII and CIV in N2a cells by inhibitor of mitochondrial translation . Mouse neuroblastoma N2a cells were treated for 120 h with chloramphenicol, which is an inhibitor of mitochondrial translation. (A) The effects of chloramphenicol treatment for the protein synthesis of components were examined. 5 mug/lane of whole-membrane protein of N2a cells with (+) or without (-) chloramphenicol treatment was separated by SDS-PAGE, and the components of complexes II, III and IV were detected by western blotting using anti-SDHA (A-1), anti-Core I (A-2) and anti-COX I antibodies (A-3). Then, anti-COX IV was reprobed onto the anti-SDHA-probed membrane without the stripping process, and complex IV was detected (A-4). Each arrow indicates SDHA (A-1), Core I (A-2), COX I (A-3) and COX IV (A-4). Asterisk indicates unstripping SDHA signals on the PVDF membrane (A-4). (B,C) 15 mug/lane of whole-membrane protein of N2a cells with (+) or without (-) chloramphenicol treatment were separated by hrCN-PAGE in a 3-14% gradient gel. E1-E4 denote signals detected by western blotting (B) and ECL solution (C). CIII (anti-Core I, B-1) and CIV (anti-COX I, B-2) were detected by western blotting with exposure times of 58 and 17 s, respectively. E1-E4 are shown as signals detected at the same position with the molecular mass. Peroxidase activity was assayed by the ECL solution after pre-incubation of 1% of SDS solution (C). CII, complex II; CIII, comple

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Mitochondrial alterations in EBS-MD muscle. a Skeletal muscle specimens from a healthy control and EBS-MD patients were histologically double-stained for SDH and COX. Note the presence of rubbed-out areas ( arrows ), and the presence of COX-negative fibers in patient 3 ( arrowheads ). Scale bar: 50 mum. b Confocal imaging of mitochondrial respiratory complex IV-stained skeletal muscle specimens from a healthy control and EBS-MD patients. Panels i-iv are magnifications of the boxed areas in panel b. Note the reduced staining intensity in all EBS-MD samples. Scale bars: 50 mum ( b ), 25 mum (panels i-iv). c Immunoblotting of cell lysates prepared from EBS-MD patients and three healthy controls using antibodies for mitochondrial respiratory complex II or V. alpha-Actinin was used as loading control. d Signal intensities of respiratory complex II or V protein bands as shown in ( c ) were densitometrically measured and normalized to the total protein content (assessed by alpha-actinin staining). Healthy controls ( dashed line ) are set to 100 %. Mean values +- SEM, 3 replicates

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Loss of YME1L in the nervous system causes microphthalmia, cataracts, and retinal inflammation A Representative images of eyes and lenses from 6- to 7-week-old wild-type (WT) and nervous system-specific YME1L knockout (NYKO) mice. Orange dashed lines mark eye morphology. Scale bars, 5 mm. B Retinal sagittal cross sections from 6- to 7-week-old mice stained with hematoxylin and eosin. NFL = nerve fiber layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer, R&C = rods and cones. Scale bars, 30 mum. C Quantification of nuclei in OPL (area = 1,000 mum 2 ) from retina cross sections of 6- to 7-week-old WT ( n = 4) and NYKO ( n = 4) mice. D Immunoblot analysis of retinal lysates from 6- to 7-week-old WT and NYKO mice. GFAP was used as a marker for reactive astrogliosis and SDHA as a loading control. E mRNA levels of proinflammatory cytokines and NF-kappaB target genes from 6- to 7-week-old retinas (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels. F mRNA levels of Fgf21 from 6- to 7-week-old retinas (WT, n = 5; NYKO, n = 5). Transcript levels were normalized to Hprt mRNA levels. G Transmission electron micrographs of optic nerves from 6- to 7-week-old WT and NYKO mice. Scale bars, 2 mum. Data information: Data were analyzed using unpaired t -test, * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure EV4 Ablation of Oma1 in NYKO mice does not restore retinal organization and causes axonal degeneration in ventro-lateral and ventral tracts of the spinal cord A Immunoblot analysis of spinal cord lysates from 6- to 7-week-old WT, NYKO, NOKO, and NYOKO mice ( n = 3) using the indicated antibodies. SDHA was used as a loading control. * indicates unspecific antibody binding. B, C Retinal sagittal cross sections from 6- to 7-week-old NYOKO mice ( n = 4) stained with hematoxylin and eosin. NFL = nerve fiber layer, IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer, R&C = rods and cones. Scale bar, 30 mum. D Transverse semithin sections of spinal cords of 6- to 7-week-old WT, NOKO, and NYOKO mice. Sections were stained with toluidine blue. Orange arrows point to MDBs indicating degenerating neurons. Scale bars, 25 mum. E MDBs in ventro-lateral and ventral tracts of 6- to 7-week-old WT ( n = 3), NOKO ( n = 3), and NYOKO ( n = 3) mice. F mRNA levels of proinflammatory cytokines from 6- to 7-week-old retinas (WT, n = 5; NYOKO, n = 5). Transcript levels were normalized to Hprt mRNA levels. Data information: Unpaired t -test was used for comparison of two groups, ordinary one-way ANOVA for comparison of three groups. ** P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 Defective proteostasis and accelerated OPA1 processing in brain and spinal cord of NYKO mice A TEM analysis of sagittal spinal cord sections of 6- to 7-week-old WT and NYKO mice. Scale bars, 500 nm. B Frequency distribution of mitochondrial lengths in spinal cords of 6- to 7-week-old WT (182 mitochondria) and NYKO mice (192 mitochondria). Kruskal-Wallis test, * P < 0.05. C TEM analysis of cerebellar sections of 28- to 32-week-old WT and NYKO mice. Scale bars, 1 mum. D Frequency distribution of mitochondrial aspect ratios were calculated from WT ( n = 210) and NYKO ( n = 230) mitochondria. Mann-Whitney test, **** P < 0.0001. Data are presented as median values. E Immunoblot analysis of forebrain, cerebellar and spinal cord lysates from 31- to 32-week-old WT and NYKO mice ( n = 3) using the indicated antibodies. SDHA and LONP1 were used to control for gel loading. * indicates unspecific antibody binding. Source data are available online for this figure.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 8 Loss of OMA1 does not restore ocular dysfunction and deteriorates axonal degeneration in the spinal cord of NYKO mice A Immunoblot analysis of retinal and spinal cord lysates from 6- to 7-week-old WT, NYKO, NOKO, and NYOKO mice ( n = 3) using the indicated antibodies. SDHA was used as a loading control. * was not reproducibly detectable in all experiments. B Frequency distribution of mitochondrial lengths in spinal cords of 6- to 7-week-old NYOKO mice (176 mitochondria) compared to WT (as in Fig 7 B). Kruskal-Wallis test, ns = not significant. C Representative images of eyes and lenses from 6- to 7-week-old WT and mice lacking OMA1 (NOKO) or both YME1L and OMA1 (NYOKO) in the nervous system. Orange dashed lines mark eye morphology. Scale bars, 2.5 mm. D Immunoblot analysis of retina lysates of 6- to 7-week-old WT, NYKO, NOKO, and NYOKO mice ( n = 3). SDHA was used to control for gel loading. E Transverse semithin sections of spinal cords of 6- to 7-week-old WT, NOKO, and NYOKO mice. Sections were stained with toluidine blue. Orange arrows point to MDBs indicating degenerating neurons. Scale bars, 25 mum. F MDBs in dorso-lateral tracts of 6- to 7-week-old WT ( n = 3), NOKO ( n = 3), and NYOKO ( n = 3) mice. G Walking beam test of 6- to 7-week-old WT ( n = 6), NOKO ( n = 5), and NYOKO ( n = 7) mice. Data information: Data were analyzed using one-way ANOVA. *** P