Journal: Nature Communications

Article Title: Spatial-fluxomics provides a subcellular-compartmentalized view of reductive glutamine metabolism in cancer cells

doi: 10.1038/s41467-019-09352-1

Figure Lengend Snippet: An approach for quantifying mitochondrial and cytosolic metabolite levels. a Rapid cell fractionation effectively removes cytosolic components (green) without damaging mitochondrial membrane. Mitochondria retain MitoTracker (red) after digitonin-based cell fractionation. While nucleus (blue) remains intact within the mitochondrial fraction, it should not bias the mitochondrial metabolite pool size measurements due to the free diffusion of small molecules through nuclear pore complexes into the cytosolic fraction. b The purity of the mitochondria and cytosol-enriched fractions in terms of the expression of mitochondrial (citrate synthase; CS, red) and cytosolic marker proteins (glyceraldehyde-phosphate dehydrogenase; GAPDH, green) via quantitative western blot (three biological replicates are shown). c – d The extent of cross-contamination between the mitochondrial and cytosolic fractions based on mitochondrial and cytosolic protein (western blot, green) and small-molecule markers (LC-MS, orange). Tetramethylrhodamine methyl ester (TMRM) was introduced as a mitochondria-specific small-molecule marker. e A comparison of the sum of measured metabolite pools in the two subcellular fractions to that measured in whole-cell extracts; a cumulative distribution shows the number of metabolites ( y -axis) for which the sum of pool sizes in the two fractions deviates by different extent from the whole-cell measurements ( x -axis). f Delay in the quenching of metabolism in the mitochondrial fraction (by 1, 5, 10, and 30 min) leads to reduced pool sizes due to the diffusion of small molecules out of mitochondria; a cumulative distribution shows the number of metabolites ( y -axis) for which the fractional pool size in the mitochondrial fraction drops by different extent ( x -axis). g The deconvolution function used to infer metabolite pool size ratio in mitochondria versus cytosol ( y -axis) given the measured pool size ratio ( x -axis), considering the cross-contamination between the two fractions. Confidence intervals of the deconvoluted pool size rations are shown considering 5% (orange) and 10% (green) coefficient of variance (CV) in the measured pool sizes in the mitochondrial and cytosolic fractions. h The ratio of metabolite pool sizes in mitochondria versus cytosol for 42 metabolites in HeLa cells under standard normoxic condition; glycolytic and pentose phosphate pathway metabolites (orange) and TCA cycle metabolites (green). Data are mean ± SD, n = 3 independent biological replicates

Article Snippet: Mitochondria were prepared with the magnetic beads method (Mitochondria Isolation Kit; Miltenyi Biotec,), and the resulting mitochondrial pellets were reconstituted in assay buffer (125 mM KCl, 10 mM Tris/MOPS, 0.1 mM EGTA/Tris, 1 mM Pi, pH 7.4) supplied with indicated nutrients and tracer .

Techniques: Cell Fractionation, Membrane, Diffusion-based Assay, Expressing, Marker, Western Blot, Liquid Chromatography with Mass Spectroscopy, Comparison

Journal: Nature Communications

Article Title: Spatial-fluxomics provides a subcellular-compartmentalized view of reductive glutamine metabolism in cancer cells

doi: 10.1038/s41467-019-09352-1

Figure Lengend Snippet: Compartment-specific isotope tracing in mitochondria and cytosol. a A schematic description of the spatial-fluxomics approach. b TCA cycle metabolism and associated reactions involving acetyl-CoA and fatty acid metabolism. c Isotopic labeling kinetics of citrate m + 2 in mitochondria versus cytosol when feeding HeLa cells with [U- 13 C]-glucose; showing the ratio between the relative abundance of the m + 2 form of citrate in the mitochondria (out of the entire mitochondrial pool) divided by the relative abundance of citrate m + 2 in cytosol. d – f The isotopic labeling kinetics of citrate m + 4 ( d ), glutamine m + 5 ( e ), and glutamate m + 5 ( f ) when feeding [U- 13 C]-glutamine. Data are mean ± SD, n = 3 independent biological replicates

Article Snippet: Mitochondria were prepared with the magnetic beads method (Mitochondria Isolation Kit; Miltenyi Biotec,), and the resulting mitochondrial pellets were reconstituted in assay buffer (125 mM KCl, 10 mM Tris/MOPS, 0.1 mM EGTA/Tris, 1 mM Pi, pH 7.4) supplied with indicated nutrients and tracer .

Techniques: Isotopic Labeling

Journal: Nature Communications

Article Title: Spatial-fluxomics provides a subcellular-compartmentalized view of reductive glutamine metabolism in cancer cells

doi: 10.1038/s41467-019-09352-1

Figure Lengend Snippet: A quantitative view of mitochondrial and cytosolic fluxes in the TCA cycle and citrate metabolism under normoxia. a – d Mass-isotopomer labeling kinetics of citrate in mitochondria ( a ) and cytosol ( b ), and malate in mitochondria ( c ) and cytosol ( d ) when feeding HeLa cells with [U- 13 C]-glutamine under standard normoxic conditions. e Measured isotopic labeling ratio in HeLa cells for citrate m + 5 /m + 4 and malate m + 3 /m + 4 in media (red) in comparison to the expected labeling via computational simulation, considering the measured labeling kinetics of citrate and malate in mitochondria (green) and cytosol (orange). f Gibbs free energy of mitochondrial (IDH2/3) and cytosolic (IDH1) IDH isozymes (in the oxidative direction) in HeLa cells under normoxia (green) and hypoxia (red). g Mitochondrial and cytosolic fluxes, showing percentage from citrate synthase flux (which is 0.48 mM h −1 ). Arrow represents the direction of net flux; number represents net flux in the direction of the arrow and number in parenthesis correspond to the backward flux. Confidence intervals for estimated fluxes are shown in Supplementary Data . h The measured mass-isotopomer distribution of palmitate when feeding cells with [U- 13 C]-glutamine (green) is consistent with the simulated fit (orange). For the simulation, acetyl-CoA labeling was assumed to follow a binomial distribution with a probability of 7.1% having m + 2 labeling form. i Validation of the method based on knock-down of IDH1 or IDH2 genes and following citrate isotopic labeling after feeding cells with [U- 13 C]-glutamine. Upon IDH1 knockdown, the ratios between citrate m + 5 and m + 4 in mitochondria and cytosol are similar, indicating that all reductive glutamine flux occurred in mitochondria (where citrate m + 4 is produced from malate m + 4). Meanwhile, IDH2 knockdown resulted in a higher citrate m + 5 to m + 4 ratio in cytosol, indicating that reductive IDH1 remains active. n.s. not significant. * P < 0.05 and ** P < 0.01 by two-sample t -test. Data are mean ± SD, n = 3 independent biological replicates

Article Snippet: Mitochondria were prepared with the magnetic beads method (Mitochondria Isolation Kit; Miltenyi Biotec,), and the resulting mitochondrial pellets were reconstituted in assay buffer (125 mM KCl, 10 mM Tris/MOPS, 0.1 mM EGTA/Tris, 1 mM Pi, pH 7.4) supplied with indicated nutrients and tracer .

Techniques: Labeling, Isotopic Labeling, Comparison, Biomarker Discovery, Knockdown, Produced

Journal: Nature Communications

Article Title: Spatial-fluxomics provides a subcellular-compartmentalized view of reductive glutamine metabolism in cancer cells

doi: 10.1038/s41467-019-09352-1

Figure Lengend Snippet: Metabolic rewiring of mitochondrial and cytosolic fluxes under hypoxia. a, b Mass-isotopomer labeling kinetics of citrate in mitochondria ( a ) and cytosol ( b ) when feeding HeLa cells with [U- 13 C]-glutamine under hypoxia. c Mitochondrial to cytosolic metabolite pool size ratios ( y -axis) under normoxia (green) and hypoxia (orange) in HeLa cells; average fold change in whole-cell pool size under normoxia and hypoxia is indicated by color ( x -axis). d Mitochondrial and cytosolic fluxes in HeLa cells under hypoxia, showing percentages from citrate synthase flux in normoxia. Arrow represents the direction of net flux; number represents net flux in the direction of the arrow and number in parenthesis correspond to the backward flux. Confidence intervals for estimated fluxes shown in Supplementary Data . * P < 0.05, ** P < 0.01, and *** P < 0.001 by two-sample t -test. Data are mean ± SD, n = 3 independent biological replicates

Article Snippet: Mitochondria were prepared with the magnetic beads method (Mitochondria Isolation Kit; Miltenyi Biotec,), and the resulting mitochondrial pellets were reconstituted in assay buffer (125 mM KCl, 10 mM Tris/MOPS, 0.1 mM EGTA/Tris, 1 mM Pi, pH 7.4) supplied with indicated nutrients and tracer .

Techniques: Labeling

Journal: Nature Communications

Article Title: Spatial-fluxomics provides a subcellular-compartmentalized view of reductive glutamine metabolism in cancer cells

doi: 10.1038/s41467-019-09352-1

Figure Lengend Snippet: Reversed CS flux supports cell survival and growth in SDH-deficient cells. a , b Isotopic labeling kinetics of cytosolic and mitochondrial citrate m + 5 and mitochondrial malate m + 3 when feeding SDH-WT ( a ) and SDH-KO cells ( b ) with [U- 13 C]-glutamine. c Mitochondrial to cytosolic metabolite pool size ratios ( y -axis) in SDH-WT (green) and SDH-KO cells (orange); average fold change in whole-cell pool size upon SDH deficiency is indicated by color ( x -axis). d Gibbs free energy of mitochondrial (IDH2/3) and cytosolic (IDH1) IDH isozymes (in the oxidative direction) in SDH-proficient (green) and deficient cells (orange). e Mitochondrial and cytosolic fluxes in SDH-KO cells, showing percentages from citrate synthase flux in the SDH-positive cells. Arrow represents the direction of net flux; number represents net flux in the direction of the arrow and number in parenthesis correspond to the backward flux. Confidence intervals for estimated fluxes shown in Supplementary Data 8. f Protein expression of mitochondrial and cytosolic markers in isolated mitochondria from SDH-WT and KO cells. g Fractional labeling of acetyl-CoA (m + 2) and malate (m + 4) in isolated mitochondria cultured with [U- 13 C]-citrate. h Fractional labeling of acetyl-CoA (m + 2) and malate (m + 4) in isolated SDH-KO mitochondria cultured with [U- 13 C]-citrate with or without ATP and ACLY inhibitor (BMS-303141). i Relative cell viability for SDH-WT (green) and SDH-KO cells (orange) following the addition of increasing concentration of dichloroacetate (DCA). XTT cell viability assay was performed 72 h after DCA treatment. j Relative metabolite pool sizes in SDH-WT (green) and SDH-KO cells (orange) with 10 mM DCA (24 h); compared to untreated control. k Metabolite fractional labeling from [U- 13 C]-glutamine in SDH-KO cells without (green) or with (orange) 10 mM DCA (24 h). * P < 0.05, ** P < 0.01, and *** P < 0.001 by multiple t -test analysis with FDR correction ( f ) or by two-sample t -test ( c , e , g , and h ). n.d. not detected. Data are mean ± SD, n = 3 independent biological replicates

Article Snippet: Mitochondria were prepared with the magnetic beads method (Mitochondria Isolation Kit; Miltenyi Biotec,), and the resulting mitochondrial pellets were reconstituted in assay buffer (125 mM KCl, 10 mM Tris/MOPS, 0.1 mM EGTA/Tris, 1 mM Pi, pH 7.4) supplied with indicated nutrients and tracer .

Techniques: Isotopic Labeling, Expressing, Isolation, Labeling, Cell Culture, Concentration Assay, Viability Assay, Control

Journal: Cell death and differentiation

Article Title: Apoptosome inactivation rescues proneural and neural cells from neurodegeneration.

doi: 10.1038/sj.cdd.4401476

Figure Lengend Snippet: Figure 1 Apaf1-deficiency leads to accumulation of NPCs expressing neural markers in vivo and in vitro. (a) BrdU staining on coronal sections from e12.5 wt and Apaf1/ (/) littermates embryos. nt: neural tube (hindbrain), fb: forebrain. (b) Analysis of differentiation markers from e14 wt and Apaf1/ littermate embryos. nestin, delta1, notch1 mRNA levels were revealed by in situ mRNA hybridization as previously described (Stoykova and Gruss, 1994) on embryonic coronal sections. Class III b- tubulin was revealed by immunohistochemistry on embryonic e14.5 coronal sections with a mouse monoclonal anti-class III b-tubulin antibody. The corresponding histological structures are indicated in the sketch and in the right panel as follows: I: intermediate zone, M: marginal zone, V: ventricular germinal zone, ve: brain ventricle. Scale bar: 0.4 mm. (c) ETNA þ / þ ( þ / þ ) and ETNA/ (/) cell lines were isolated from e14 wt and mutant embryos, respectively, and immortalized with a retrovirus transducing the tsA58/U19 large T antigen. Total extracts from both cell lines were immunoprecipitated with an anti-Apaf1 rabbit polyclonal antibody and analysed with a rat monoclonal antibody anti-Apaf1. Ctrl: human neuroblastoma cells (SH-SY5Y) stably overexpressing Apaf1. (d) Total cell lysates from ETNA þ / þ and ETNA/ cells, either naı¨ve (N) or incubated for 48 h with a differentiation medium (D), were analysed by Western blotting for the expression of NeuN, class III b-tubulin, ChAT, PSD95, tau; b-actin was also assayed as a control of equal protein loading. (e) ETNA þ / þ and ETNA/ cells, either naı¨ve or differentiated, were immunostained with an antibody recognizing the 160 kDa form of neurofilament (NF-M). Scale bar: 40 mm

Article Snippet: We used an anti-cytochrome c mouse monoclonal antibody (clone 6H2.B4, BD PharMingen), an anti-cytochrome c rabbit polyclonal antibody (Santa Cruz), an anti-AIF rabbit polyclonal antibody (a generous gift of Guido Kroemer), an anti-EndoG rabbit polyclonal antibody (ProSci Incorporated), an anti-Neurofilament 160 mouse monoclonal antibody (clone NN18, SIGMA), anti-Apaf1 polyclonal antibody (AB16941, Chemicon Int.) and an anti- Large T Antigen monoclonal antibody (clone PAb419, Oncogene).

Techniques: Expressing, In Vivo, In Vitro, BrdU Staining, In Situ, Hybridization, Immunohistochemistry, Isolation, Mutagenesis, Immunoprecipitation, Stable Transfection, Incubation, Western Blot, Control

Journal: Frontiers in Pediatrics

Article Title: Case Report: A Novel Intronic Mutation in AIFM1 Associated With Fatal Encephalomyopathy and Mitochondrial Disease in Infant

doi: 10.3389/fped.2022.889089

Figure Lengend Snippet: (A) Relative quantification of mRNA levels for AIFM1 in controls and patient’s fibroblasts using different primers. The results of mRNA are the average of the values assessed after three reaction tests. (B) Western blot analysis in controls and patient’s fibroblasts using AIF antibody. GAPDH was used as loading control. The patient’s sample shows a clear reduction in the AIF amount compared with the control lines. (C) Western blot analysis in controls and patient’s muscles using AIF antibody. GAPDH was used as loading control. (D) Electron microscopy of quadriceps muscle demonstrating large, irregularly shaped mitochondria, including one with concentric cisternae. (E) TOMM20 staining of the patient’s and control fibroblast. DAPI was used to mark the nucleus.

Article Snippet: A mouse monoclonal anti-GAPDH (4A Biotech Co., Ltd., Shanghai, China, Cat. No. 4ab030004) and mouse monoclonal anti-AIFM1 antibody binding to the C-terminus of human AIF (Boster Biological Technology, Wuhan, China, Cat. No. M01571-1) were used.

Techniques: Quantitative Proteomics, Western Blot, Control, Muscles, Electron Microscopy, Staining

Journal: Frontiers in Pediatrics

Article Title: Case Report: A Novel Intronic Mutation in AIFM1 Associated With Fatal Encephalomyopathy and Mitochondrial Disease in Infant

doi: 10.3389/fped.2022.889089

Figure Lengend Snippet: (A) Schematic model representing the AIF protein. AIF is a flavoprotein (with an oxidoreductase enzymatic activity) containing a FAD-bipartite domain (amino-acids 128–262 and 401–480), a NADH-binding motif (amino-acids 263–400), and a C-terminal domain (amino-acids 481–608) where the proapoptotic activity of the protein resides. In addition, it has a Mitochondria Localization Sequence (MLS, amino-acids 1–41) placed in its N-terminal region. AIF also possesses two DNA-binding sites, which are located in amino-acids 255–265 and 510–518, respectively. (B) The mutant causes a frameshift resulted in the change of amino acid coding after R358, and the early termination codon was generated at the position of amino acid 362. (C) Protein structure modeling of wild-type and mutated AIFM1. A part of the amino acid sequence has been eliminated in the mutated protein compared to the wild type protein. (D) The NAD + /NADH ratios in fibroblasts of patient and control. (E) The human COX of cell culture supernatant in patient and control. *Statistically significant difference at p < 0.05.

Article Snippet: A mouse monoclonal anti-GAPDH (4A Biotech Co., Ltd., Shanghai, China, Cat. No. 4ab030004) and mouse monoclonal anti-AIFM1 antibody binding to the C-terminus of human AIF (Boster Biological Technology, Wuhan, China, Cat. No. M01571-1) were used.

Techniques: Activity Assay, Binding Assay, Sequencing, Mutagenesis, Generated, Control, Cell Culture