Disruption of the TCA cycle reveals an ATF4-dependent integration of redox and amino acid metabolism

  1. Dylan Gerard Ryan
  2. Ming Yang
  3. Hiran A Prag
  4. Giovanny Rodriguez Blanco
  5. Efterpi Nikitopoulou
  6. Marc Segarra-Mondejar
  7. Christopher A Powell
  8. Tim Young
  9. Nils Burger
  10. Jan Lj Miljkovic
  11. Michal Minczuk
  12. Michael P Murphy
  13. Alex von Kriegsheim
  14. Christian Frezza  Is a corresponding author
  1. MRC Cancer Unit, University of Cambridge, Hutchison MRC Research Centre, Cambridge Biomedical Campus, United Kingdom
  2. MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, United Kingdom
  3. Department of Medicine, University of Cambridge, Cambridge Biomedical Campus, United Kingdom
  4. Edinburgh Cancer Research UK Centre, Institute of Genetics and Cancer, United Kingdom
8 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
TCA cycle inhibition promotes cytosolic glutathione synthesis while impairing de novo proline and aspartate synthesis.

(A) Schematic diagram of comparative metabolomic approach to assess the metabolic response of kidney epithelial cells to TCAi. (B) Significantly increased metabolites (Cut-off = 25% change in abundance, false discovery rate (FDR) = 5%) with genetic or pharmacological TCAi. (C) Significantly decreased metabolites. (B–C) (24 hr timepoint). (D) Volcano plot of glutathione-related metabolites and amino acids in Fh1-/- versus Fh1fl/fl cells. (E) Volcano plot of glutathione-related metabolites and amino acids in Sdhb-/- versus Sdhbfl/fl cells. (F) Heatmap of mean scaled consumption-release (CoRe) intensity value of amino acids in Fh1-/- versus Fh1fl/fl cells. (B–F) (n = 5–10 independent biological replicates). (G) Volcano plot of glutathione-related metabolites and amino acids in FHIN-1 (20 μM)-treated Fh1fl/fl versus DMSO-vehicle control Fh1fl/fl cells. (H) Volcano plot of glutathione-related metabolites and amino acids in AA5 (1 μM)-treated Fh1fl/fl versus DMSO-treated Fh1fl/fl cells. (G–H) (24 hr timepoint) (n = 10–15 independent biological replicates). (I) Heatmap of mean scaled CoRe intensity value of amino acids in FHIN-1, AA5 and TTFA (500 μM) versus DMSO-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). (D–I) p Value determined by multiple unpaired t-tests, corrected with two-stage step-up method of Benjamini, Krieger, and Yekutieli - FDR = 5%. p < 0.05*; p < 0.01**; p < 0.001***.

Figure 1—figure supplement 1
Metabolomic analysis of acute and chronic TCA cycle inhibition.

(A) PCA plot of metabolomics data of Fh1fl/fl, Fh1-/-CL1, Fh1-/-CL19 and Fh1-/-CL1+pFh1 cells (n = 5 independent biological replicates). (B) PCA plot of metabolomics data of Sdhbfl/fl, Sdhb-/-CL5 and Sdhb-/-CL7 cells (n = 5 independent biological replicates). (C) PCA plot of DMSO- and FHIN-1 (20 μM)-treated Fh1fl/fl cells (24 h timepoint) (n = 5 independent biological replicates). (D) PCA plot of DMSO-, AA5 (1 μM)- and TTFA (500 μM)-treated Fh1fl/fl cells (24 h timepoint) (n = 5 independent biological replicates). (E) Heatmap of top 50 significant metabolite changes in Fh1fl/fl, Fh1-/-CL1, Fh1-/-CL19 and Fh1-/-CL1+pFh1 cells (n = 5 independent biological replicates) and relative change in Fh1-deficient metabolic markers (S)–2-succinocysteine (2SC), succinicGSH, malate and aspartate (n = 5 independent biological replicates). (F) Heatmap of top 50 significant metabolite changes in Sdhbfl/fl, Sdhb-/-CL5, and Sdhb-/-CL7 cells (n = 5 independent biological replicates) and relative change in Sdh-deficient metabolic markers succinate, fumarate, malate, and aspartate (n = 5 independent biological replicates). (G) Heatmap of top 50 significant metabolite changes in DMSO- and FHIN-1 (20 μM)-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates) and relative change in Fh1-deficient metabolic markers 2SC, succinicGSH, malate and aspartate (n = 5 independent biological replicates). (H) Heatmap of top 50 significant metabolite changes in DMSO-, AA5 (1 μM)- and TTFA (500 μM)-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates) and relative change in Sdh-deficient metabolic markers succinate, fumarate, malate, and aspartate (n = 5 independent biological replicates). (E–H) Data are mean ± SEM. p value determined by ordinary one-way ANOVA, corrected for multiple comparisons using Tukey statistical hypothesis testing. p < 0.05*; p < 0.01**; p < 0.001***. (I) Volcano plot of log2 fold change in glutathione-related metabolites and amino acids in TTFA (500 μM)-treated Fh1fl/fl versus DMSO-vehicle control Fh1fl/fl cells (24 hr timepoint) (n = 10 independent biological replicates). (J) Volcano plot of log2 fold change in glutathione-related metabolites and amino acids in FHIN-1 (20 μM)-treated Fh1fl/fl versus DMSO-vehicle control Fh1fl/fl cells (1 hr timepoint) (n = 5 independent biological replicates). (K) Volcano plot of log2 fold change glutathione-related metabolites and amino acids in AA5 (1 μM)-treated Fh1fl/fl versus DMSO-treated Fh1fl/fl cells (1 hr timepoint) (n = 5 independent biological replicates). p value determined by multiple unpaired t-tests, corrected with two-stage step-up method of Benjamini, Krieger, and Yekutieli - FDR = 5%.

Figure 2 with 1 supplement
TCA cycle inhibition impairs glutamine-derived proline and aspartate synthesis but promotes cytosolic GSH synthesis.

(A) Schematic diagram highlighting U-13C-glutamine tracing into distinct metabolic modules. (B) U-13C-glutamine tracing into proline (m + 5 labeling intensity and total isotopologue fraction distribution). (C) U-13C-glutamine tracing into aspartate, asparagine and alanine (m + 4 and m + 3 labeling intensity and total isotopologue fraction distribution). (D) U-13C-glutamine tracing into glutathione synthesis pathway (m + 5 and m + 10 labeling intensity and total isotopologue fraction distribution) (B–D) show DMSO, FHIN-1 (20 μM)-, AA5 (1 μM)- and TTFA (500 μM)-treated Fh1fl/fl cells (24 h timepoint) (n = 5 independent biological replicates). Data are mean ± standard error of mean (SEM). p Value determined by ordinary one-way ANOVA, corrected for multiple comparisons using Tukey statistical hypothesis testing. (E) Proline and aspartate level fold change in mitochondrial and cytosol fractions in FHIN-1 and AA5-treated versus DMSO-treated Fh1fl/fl cells (24 h timepoint) (n = 3 independent biological replicates). Data are mean ± standard error of mean (SEM). p Values determined by unpaired two-tailed t-test. p < 0.05*; p < 0.01**; p < 0.001***.

Figure 2—figure supplement 1
Glutamine fate tracing and compartment-specific metabolomics upon acute TCA cycle inhibition.

(A) Schematic diagram highlighting U-13C-glutamine tracing into the TCA cycle and reductive carboxylation. (B) U-13C-glutamine tracing into α-KG, succinate, fumarate, malate, citrate, cis-aconitate and 2-HG (total isotopologue fraction distribution) in DMSO, FHIN-1 (20 μM), AA5 (1 μM), and TTFA (500 μM)-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). (C) U-13C-glutamine tracing into α-KG, succinate, fumarate, malate, citrate, cis-aconitate, and 2-HG (m + 5 and m + 3 labeling intensity) in DMSO, FHIN-1 (20 μM), AA5 (1 μM) and TTFA (500 μM)-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). Data are mean ± SEM. p Value determined by ordinary one-way ANOVA, corrected for multiple comparisons using Tukey statistical hypothesis testing. p < 0.05*; p < 0.01**; p < 0.001***. (D) Glutamine, glutamate, GSH, GSSG, alanine, asparagine level fold changes and succGSH relative intensity in mitochondrial and cytosol fractions in FHIN-1 (20 μM)- and AA5(1 μM)-treated versus DMSO-treated Fh1fl/fl cells (24 hr timepoint) (n = 3 independent biological replicates). Data are mean ± SEM. p values determined by unpaired two-tailed t-test. p < 0.05*; p < 0.01**; p < 0.001***.

Figure 3 with 1 supplement
Disrupted mitochondrial thiol redox homeostasis phenocopies TCA cycle inhibition.

(A) Schematic diagram highlighting mito 1-Chloro-2,4-dinitrobenzene (mitoCDNB) reaction with glutathione and the formation of mito 1-S-glutathionyl-2,4-dinitrobenzene (MitoGSDNB) and intensity levels in DMSO-treated and mitoCDNB (10 μM)-treated Fh1fl/fl cells. (B) Volcano plot of glutathione-related metabolites and amino acids in mitoCDNB-treated Fh1fl/fl versus DMSO-treated Fh1fl/fl cells. p Value determined by multiple unpaired t-tests, corrected with two-stage step-up method of Benjamini, Krieger and Yekutieli - FDR = 5%. (C) U-13C-glutamine tracing (m + 10, m + 5, m + 4 and m + 3 labeling intensity and total isotopologue fraction distribution) in DMSO and mitoCDNB-treated Fh1fl/fl cells. (A–C) (24 hr timepoint) (n = 5–10 independent biological replicates). (A and C) Data are mean ± SEM. p Value determined by unpaired two tailed t-test or ordinary one-way ANOVA corrected for multiple comparisons using Tukey statistical hypothesis testing. p < 0.05*; p < 0.01**; p < 0.001***.

Figure 3—figure supplement 1
Glutamine fate tracing upon mitochondrial GSH depletion.

(A) Interleaved box and whiskers plot of fold change in GSH and GSSG with FHIN-1 (20 μM)-, AA5 (1 μM)- and TTFA (500 μM)-treated Fh1fl/fl cells versus DMSO-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). p values determined by unpaired two-tailed t-test. p < 0.05*; p < 0.01**; p < 0.001***. (B) U-13C-glutamine tracing into α-KG, succinate, fumarate, malate, citrate, cis-aconitate, and 2-HG (total isotopologue fraction distribution) in DMSO and mitoCDNB (10 μM)-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). (C) U-13C-glutamine tracing into α-KG, succinate, fumarate, malate, citrate, cis-aconitate, and 2-HG (m + 5 and m + 3 labeling intensity) in DMSO and mitoCDNB (10 μM)-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). (D) ATP/ADP, lactate/pyruvate and NADH/NAD+ ratio in DMSO and mitoCDNB (10 μM)-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). Data are mean ± SEM. p Values determined by unpaired two-tailed t-test. p < 0.05*; p < 0.01**; p < 0.001***.

Figure 4 with 1 supplement
Impaired mitochondrial respiration underlies defect in de novo proline synthesis.

(A) Scaled intensity values of glutathione-related metabolites in BSO (500 μM)-treated and DMSO-treated Fh1fl/fl cells (24 hr timepoint). (B) Volcano plot of amino acids in BSO-treated versus DMSO-treated Fh1fl/fl cells (24 hr timepoint). (C) Interleaved box and whiskers plot of amino acids with FHIN-1-treated or FHIN-1 and BSO co-treatment of Fh1fl/fl cells (24 hr timepoint). (D) Interleaved box and whiskers plot of amino acids with AA5-treated or AA5 and BSO co-treatment of Fh1fl/fl cells. (E) NADH/NAD+, NADPH/NADP+ and ATP/ADP ratio in DMSO, FHIN-1- and AA5-treated Fh1fl/fl cells ± BSO or ethylGSH co-treatment. (F) OCR assessed by Seahorse analyser in DMSO, FHIN-1-, AA5-, and TTFA-treated Fh1fl/fl cells (A–F) (n = 5 independent biological replicates). (E–F) Data are mean ± SEM. p Value determined by ordinary one-way ANOVA, corrected for multiple comparisons using Tukey statistical hypothesis testing. p < 0.05*; p < 0.01**; p < 0.001***. (G) NADH/NAD+, NADPH/NADP+, and ATP/ADP ratio in DMSO and Oligomycin (10 μM)-treated Fh1fl/fl cells (1 hr timepoint). Data are mean ± SEM. p Values determined by unpaired two-tailed t-test. p < 0.05*; p < 0.01**; p < 0.001***. (H) Volcano plot of glutathione-related metabolites and amino acids in Oligomycin-treated versus DMSO-treated Fh1fl/fl cells (1 h timepoint) (G–H) (n = 5 independent biological replicates). (I) Volcano plot of glutathione-related metabolites and amino acids in Antimycin A (2 μM)-treated versus DMSO-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). (A-D; H–I) p Value determined by multiple unpaired t-tests, corrected with two-stage step-up method of Benjamini, Krieger, and Yekutieli - FDR = 5%.

Figure 4—figure supplement 1
eGSH supplementation upon acute TCA cycle inhibition.

(A) Volcano plot of log2 fold change in glutathione-related metabolites and amino acids in eGSH (1 mM)-treated versus DMSO-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). (B) Interleaved box and whiskers plot of fold change in amino acids with FHIN-1 (20 μM)-treated or FHIN-1 (20 μM) and eGSH (1 mM) co-treatment of Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). (C) Interleaved box and whiskers plot of fold change in amino acids with AA5 (1 μM)-treated or AA5 (1 μM) and eGSH (1 mM) co-treatment of Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). (A–C) p Value determined by multiple unpaired t-tests, corrected with two-stage step-up method of Benjamini, Krieger, and Yekutieli - FDR = 5%. p < 0.05*; p < 0.01**; p < 0.001***. (D) Lactate/pyruvate and citrate/pyruvate ratio in DMSO-, FHIN-1 (20 μM)- and AA5 (1 μM)-treated Fh1fl/fl cells (24 hr timepoint) (n = 15 independent biological replicates). Data are mean ± SEM. p Values determined by unpaired two-tailed t-test. p < 0.05*; p < 0.01**; p < 0.001***.

Figure 5 with 1 supplement
TCA cycle inhibition mimics an amino acid deprivation response and promotes compensatory reprogramming.

(A) GSEA of RNAseq data from FHIN-1- and AA5-treated versus DMSO-treated Fh1fl/fl cells. (B) Quantitative PCR results from FHIN-1-, AA5-, TTFA-, and DMSO-treated Fh1fl/fl cells (24 hr timepoint) (n = 3–5 independent biological replicates). Data are mean ± SEM. p Value determined by unpaired two-tailed t-test or ordinary one-way ANOVA, corrected for multiple comparisons using Tukey statistical hypothesis testing. p < 0.05*; p < 0.01**; p < 0.001***. (C) ORA of overlapping transcripts significantly increased in FHIN-1 and AA5-treated Fh1fl/fl cells and between overlapping proteins significantly increased in FHIN-1 and TTFA-treated Fh1fl/fl cells (24 h timepoint) (n = 3–5 independent biological replicates). Reactome pathways ranked by p value. (D) Comparison of amino acid metabolism and tRNA synthetase transcript and protein levels.

Figure 5—figure supplement 1
Over representation analysis of significantly decreased targets upon acute TCA cycle inhibition.

(A) ORA of overlapping transcripts significantly decreased in FHIN-1 (20 μM)- and AA5 (1 μM)-treated Fh1fl/fl cells (24 h timepoint) (24 h timepoint) (n = 3 independent biological replicates). (B) ORA of overlapping proteins significantly decreased in FHIN-1 and TTFA-treated Fh1fl/fl cells (24 h timepoint) (n = 5 independent biological replicates). (C) ORA of proteins significantly decreased uniquely in FHIN-1-treated Fh1fl/fl cells (24 h timepoint) (n = 5 independent biological replicates).(A–C) Reactome pathways ranked by combined score (p value and Z score). (D) 35S-methionine labeling of mitochondrial translation in DMSO-, FHIN-1 (20 μM)-, and AA5 (1 μM)-treated Fh1fl/fl cells (24 hr timepoint) (n = 3 independent biological replicates).

Figure 6 with 1 supplement
TCA inhibition activates the integrated stress response to counter amino acid and redox stress.

(A) Clustergram showing Transcription-factor (TF)-gene co-occurrence ORA of overlapping transcripts significantly increased in FHIN-1 and AA5-treated Fh1fl/fl cells. (B) Clustergram showing TF-gene co-occurrence ORA of overlapping transcripts significantly increased in FHIN-1 and TTFA-treated Fh1fl/fl cells. (A–B) (24 hr timepoint) (n = 3–5 independent biological replicates). TFs are ranked by combined score (p value and Z score). (C) Western blot analysis of puromycin incorporation, Atf4, phopho-eIF2α and β-actin as loading control in FHIN-1, AA5 and TTFA timecourse treatment of Fh1fl/fl cells (representative image of three independent biological replicates). (D) Western blot analysis of Atf4 and β-actin as loading control (6 hr timepoint) and qPCR analysis of Atf4-target genes (24 hr timepoint) (n = 3 independent biological replicates) in DMSO-, ISRIB (500 nM)-, FHIN-1 (10 μM), and FHIN-1+ ISRIB treated Fh1fl/fl cells. Data are mean ± SEM. p value determined by ordinary one-way ANOVA, corrected for multiple comparisons using Tukey statistical hypothesis testing. (E) Interleaved box and whiskers plot of amino acids with FHIN-1-treated or FHIN-1- and ISRIB- co-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). p Value determined by multiple unpaired t-tests, corrected with two-stage step-up method of Benjamini, Krieger, and Yekutieli - FDR = 5%. p < 0.05*; p < 0.01**; p < 0.001***.

Figure 6—figure supplement 1
Atf4 activation upon acute TCA cycle inhibition and mitochondrial GSH depletion.

(A) GSEA of Atf4 gene signature queried with molecular signature database (MsigDB) hallmarks geneset on RNA sequencing data from FHIN-1 (20 μM)- versus DMSO-treated Fh1fl/fl cells (24 hr timepoint) (n = 3 independent biological replicates). (B) GSEA of Atf4 gene signature queried with MsigDB hallmarks geneset on RNA sequencing data from AA5 (1 μM)- versus DMSO-treated Fh1fl/fl cells (24 hr timepoint) (n = 3 independent biological replicates). (C) Western blot analysis and quantification of Atf4 and β-actin as loading control in DMSO and mitoCDNB (10 μM)-treated Fh1fl/fl cells (6 hr timepoint) (n = 3 independent biological replicates). Data are mean ± SEM. p Values determined by unpaired two-tailed t-test. p < 0.05*; p < 0.01**; p < 0.001***. (D) Volcano plot of log2 fold change in glutathione-related metabolites and amino acids in ISRIB (500 nM)-treated versus DMSO-treated Fh1fl/fl cells (24 hr timepoint) (n = 5 independent biological replicates). (E) Interleaved box and whiskers plot of fold change in GSH and GSSG in FHIN-1 (10 μM)-treated or FHIN-1 and ISRIB co-treated Fh1fl/fl cells and the ratio of the fold change (24 hr timepoint) (n = 5 independent biological replicates). p Values determined by unpaired two-tailed t-test. p < 0.05*; p < 0.01**; p < 0.001***.

Figure 7 with 1 supplement
Atf4 regulates amino acid and GSH metabolism.

(A) Western blot analysis of Atf4 and β-actin as loading control (24 hr timepoint) (representative image of 3 independent biological replicates) and qPCR analysis of Atf4-target genes (24 hr timepoint) (n = 3 independent biological replicates) in DMSO-, FHIN-1- and AA5-treated Fh1fl/fl cells with or without siRNA-mediated silencing of Atf4. Data are mean ± SEM. p value determined by ordinary one-way ANOVA, corrected for multiple comparisons using Tukey statistical hypothesis testing. p < 0.05*; p < 0.01**; p < 0.001***. (B) Volcano plot of glutathione-related metabolites and amino acids in Atf4-silenced Fh1fl/fl cells versus non-targeting control (NC)- Fh1fl/fl cells (48 hr timepoint). (C) Heatmap of amino acid levels in FHIN-1- and AA5- versus DMSO-treated Fh1fl/fl cells with or without siRNA-mediated silencing of Atf4 (24 hr timepoint). (B–C) (n = 5 independent biological replicates). (D) Schematic diagram highlighting key findings of the comparative analysis between acute FHi versus SDHi.

Figure 7—figure supplement 1
Changes in glutathione metabolism intermediates upon Atf4 silencing and acute TCA cycle inhibition.

(A) Interleaved box and whiskers plot of fold change in glutathione-related metabolites in DMSO- versus FHIN-1 (20 μM)-treated Fh1fl/fl cells with or without siRNA-mediated silencing of Atf4 (24 hr timepoint) (n = 5 independent biological replicates). (B) Interleaved box and whiskers plot of fold change in glutathione-related metabolites in DMSO- versus AA5 (1 μM)-treated Fh1fl/fl cells with or without siRNA-mediated silencing of Atf4 (24 hr timepoint) (n = 5 independent biological replicates). (A–B) p value determined by multiple unpaired t-tests, corrected with two-stage step-up method of Benjamini, Krieger and Yekutieli - FDR = 5%. p < 0.05*; p < 0.01**; p < 0.001***.

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Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Mus musculus)Fh1fl/flFrezza et al., 2011Fh1fl/flKidney epithelial cell line
Cell line (Mus musculus)Fh1-/-CL1Frezza et al., 2011Fh1-/-CL1Kidney epithelial cell line
Cell line (Mus musculus)Fh1-/-CL19Frezza et al., 2011Fh1-/-CL19Kidney epithelial cell line
Cell line (Mus musculus)Sdhbfl/flCardaci et al., 2015Sdhbfl/flKidney epithelial cell line
Cell line (Mus musculus)Sdhb-/-CL5Cardaci et al., 2015Sdhb-/-CL5Kidney epithelial cell line
Cell line (Mus musculus)Sdhb-/-CL7Cardaci et al., 2015Sdhb-/-CL7Kidney epithelial cell line
Chemical compound, drugDimethyl sulfoxide (DMSO)Sigma AldrichD8418
Chemical compound, drugFumarate hydratase inhibitor 1 (FHIN-1)MedChemExpressCat. No.: HY-100004
Chemical compound, drugAtpenin A5 (AA5)AbcamCAS Number: 119509-24-9
Chemical compound, drugThenoyltrifluoroacetone (TTFA)Sigma AldrichT27006
Chemical compound, drugL-Buthionine-sulfoximine (BSO)Sigma AldrichB2515
Chemical compound, drugethylGSH (eGSH)Sigma AldrichG1404
Chemical compound, drugOligomycin ASigma Aldrich75,351
Chemical compound, drugPuromycin dihydrochlorideSigma AldrichP9620
Chemical compound, drugAntimycin ASigma AldrichA8674
Chemical compound, drugCarbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP)Sigma AldrichC2920
Chemical compound, drugRotenoneSigma AldrichR8875
Chemical compound, drugIntegrated stress response inhibitor (ISRIB)Sigma AldrichSML0843
Chemical compound, drugGlucoseSigma AldrichG8270
Chemical compound, drugGlutamineSigma AldrichG7513
Chemical compound, drugPyruvateSigma AldrichP2256
Chemical compound, drugU-13C-GlutamineCambridge IsotopesCLM-1822-H-PK
Chemical compound, drug35S-methioninePerkinElmerNEG009L005MC
Chemical compound, drugmitoCDNBBooty et al., 2019SML2573**Available on Sigma
Antibodypuromycin antibody clone 12D10 “(mouse monoclonal)”Sigma AldrichMABE343“(1:10,000)”
Antibodyanti-Atf4 (D4B8) “(rabbit monoclonal)”Cell Signalling TechnologyRabbit mAb #11,815“(1:800)”
Antibodyanti-p-eIF2α (Ser51) (D9G8) “(rabbit monoclonal)”Cell Signalling TechnologyXP Rabbit mAb #3,398“(1:1000)”
Antibodyanti-β-Actin (13E5) “(rabbit monoclonal)”Cell Signalling TechnologyRabbit mAb #4,970“(1:1000)”
Chemical compound, drugU-13C-GlutamineCambridge IsotopesCLM-1822-H-PK
Chemical compound, drug35S-methioninePerkinElmerNEG009L005MC
Transfected construct (Mus musculus)Silencer select Non-targeting control (NTC) siRNAThermo ScientificCatalog # 4390843
Transfected construct (Mus musculus)Silencer select Atf4 siRNA #1Thermo ScientificCatalog # 4390771 Assay ID s62691
Transfected construct (Mus musculus)Silencer selectAtf4 siRNA #2Thermo ScientificCatalog # 4390771 Assay ID s62690
Transfected construct (Mus musculus)Lipofectamine RNAiMAXThermo ScientificCatalog # 13778075

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  1. Dylan Gerard Ryan
  2. Ming Yang
  3. Hiran A Prag
  4. Giovanny Rodriguez Blanco
  5. Efterpi Nikitopoulou
  6. Marc Segarra-Mondejar
  7. Christopher A Powell
  8. Tim Young
  9. Nils Burger
  10. Jan Lj Miljkovic
  11. Michal Minczuk
  12. Michael P Murphy
  13. Alex von Kriegsheim
  14. Christian Frezza
(2021)
Disruption of the TCA cycle reveals an ATF4-dependent integration of redox and amino acid metabolism
eLife 10:e72593.
https://doi.org/10.7554/eLife.72593