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Mitochondrial ubiquinol oxidation is necessary for tumour growth

Abstract

The mitochondrial electron transport chain (ETC) is necessary for tumour growth1,2,3,4,5,6 and its inhibition has demonstrated anti-tumour efficacy in combination with targeted therapies7,8,9. Furthermore, human brain and lung tumours display robust glucose oxidation by mitochondria10,11. However, it is unclear why a functional ETC is necessary for tumour growth in vivo. ETC function is coupled to the generation of ATP—that is, oxidative phosphorylation and the production of metabolites by the tricarboxylic acid (TCA) cycle. Mitochondrial complexes I and II donate electrons to ubiquinone, resulting in the generation of ubiquinol and the regeneration of the NAD+ and FAD cofactors, and complex III oxidizes ubiquinol back to ubiquinone, which also serves as an electron acceptor for dihydroorotate dehydrogenase (DHODH)—an enzyme necessary for de novo pyrimidine synthesis. Here we show impaired tumour growth in cancer cells that lack mitochondrial complex III. This phenotype was rescued by ectopic expression of Ciona intestinalis alternative oxidase (AOX)12, which also oxidizes ubiquinol to ubiquinone. Loss of mitochondrial complex I, II or DHODH diminished the tumour growth of AOX-expressing cancer cells deficient in mitochondrial complex III, which highlights the necessity of ubiquinone as an electron acceptor for tumour growth. Cancer cells that lack mitochondrial complex III but can regenerate NAD+ by expression of the NADH oxidase from Lactobacillus brevis (LbNOX)13 targeted to the mitochondria or cytosol were still unable to grow tumours. This suggests that regeneration of NAD+ is not sufficient to drive tumour growth in vivo. Collectively, our findings indicate that tumour growth requires the ETC to oxidize ubiquinol, which is essential to drive the oxidative TCA cycle and DHODH activity.

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Fig. 1: Complex III is necessary for tumour growth.
Fig. 2: Ubiquinol oxidation by complex III is necessary for tumour growth.
Fig. 3: Complex I is necessary for tumour growth.
Fig. 4: Mitochondrial NAD+ regeneration is necessary but not sufficient for tumour growth in vivo.

Data availability

All data from the manuscript are available from the corresponding author on request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the NIH (5R35CA197532) to N.S.C.; a postdoctoral fellowship by Ramon Areces Foundation of Spain to I.M.R.; NCI (T32 CA009560) and NIH (2T32HL076139-16) to H. Kong; NIH (5 T32 CA 9560-33) and Northwestern University Pulmonary department’s Cugell fellowship to K.V.; NIH (T32CA09560 and T32HL076139-13) to G.S.M., and NIH (T32 T32HL076139) to S.E.W. E.M.S. is a Cancer Research Institute Irvington Fellow supported by the Cancer Research Institute. We thank Robert H. Lurie Cancer Center Flow Cytometry facility supported by NCI CCSG P30 CA060553 for their invaluable assistance. Imaging work was performed at the Northwestern University Center for Advanced Microscopy generously supported by NCI CCSG P30 CA060553 awarded to the Robert H Lurie Comprehensive Cancer Center. We thank T. Papagiannakopoulos and S. Leboeuf for providing KP cells. We thank C. Moraes for CYTB-Δ cells. Original LbNOX constructs were generated by V. Mootha’s laboratory and acquired from Addgene. We thank E. Dufour and H. Jacobs for original NDI1 and AOX constructs. We thank P. Ntziachristos for the MIGR1-Notch1ΔE-GFP vector. We thank the members of the Chandel laboratory for discussions.

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Authors

Contributions

I.M.-R. and N.S.C. conceptualized the study, interpreted the data, and wrote the manuscript with the input of co-authors. I.M.-R., L.R.C., H. Kong, M.W., H. Kihshen, G.S.M., C.R.R. and K.V. carried out most of the experiments in the paper. I.M.-R., L.R.C. and H. Kong performed leukaemia experiments. I.M.-R., L.R.C., H. Kong and K.V. performed lung tumour experiments. I.M.-R., L.R.C., C.R.R, G.S.M. and S.E.W. carried out xenograft experiments. I.M.-R., M.W. and H. Kihshen performed experiments to analyse the OCR in intact and permeabilized cells. I.M.-R., C.R.R. and M.W performed experiments to assess proliferation. I.M.-R. and M.W. performed western blot experiments. I.M.-R., L.R.C., G.S.M. and P.G. conducted and analysed metabolomics and carbon flux experiments. R.P., G.R.S.B., E.M.S. and S.E.W. provided technical expertise with mouse experiments.

Corresponding author

Correspondence to Navdeep S. Chandel.

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N.S.C. is a scientific advisory board member of Rafael Pharmaceuticals.

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Extended data figures and tables

Extended Data Fig. 1 Metabolite changes in complex III deficient cells in the presence or absence of pyruvate and uridine.

a, b, Schematic representation of the ETC in 143B-CYTB-WT (a) and 143B-CYTB-Δ cells (b). c, Coupled OCR of 143B-CYTB-WT and 143B-CYTB-Δ cells (n = 5 biologically independent experiments). d, 143B-CYTB-WT and 143B-CYTB-Δ cells were grown in the presence or absence of methyl pyruvate and/or uridine and cell number was assessed after 72 h (n = 5 biologically independent experiments). e, Intracellular aspartate levels in the presence of methyl pyruvate and uridine in 143B-CYTB-WT and 143B-CYTB-Δ cells (n = 4 biologically independent experiments). f, Intracellular aspartate levels in the absence of methyl pyruvate and uridine in 143B-CYTB-WT and 143B-CYTB-Δ cells (n = 5 biologically independent experiments). g, Intracellular NAD+/NADH ratio in the absence of methyl pyruvate and uridine of 143B-CYTB-WT and 143B-CYTB-Δ cells (n = 5 biologically independent experiments). h, The heat map displays the relative abundance of significantly changed metabolites in 143B-CYTB-WT, 143B-CYTB-Δ cells and in 143B-CYTB-Δ cells expressing either GFP or AOX in the absence of methyl pyruvate and uridine. A red–blue colour scale depicts the abundance of the metabolites (red: high, blue: low) (n = 5 biologically independent experiments). i, The heat map displays the relative abundance of significantly changed metabolites in 143B-CYTB-WT and 143B-CYTB-Δ cells in the presence of methyl pyruvate and uridine (n = 4 biologically independent experiments). j, Tumour mass of xenografts from 143B-CYTB-WT and 143B-CYTB-Δ cells (n = 10 mice per group from two independent cohorts). k, Coupled OCR of KP-NT and KP-QPC_KO cells (n = 10 technical replicates from two independent experiments). Data are mean ± s.e.m. (cg, j) or mean ± s.d. (k). *P < 0.05, **P < 0.01, two-tailed t-tests (c, g, j), two-way ANOVA (d) with a Bonferroni test for multiple comparisons or one-way ANOVA (k) with a Bonferroni test for multiple comparisons (exact P values are in the Source Data). Metabolites levels were analysed with multiple one-way ANOVA using an FDR of 0.1 and Fisher’s least significant difference test post hoc analyses Q = 10%. For two-group heat maps, t-tests with an FDR cut-off value of 0.1 were used to identify significantly changed metabolites. Each row was analysed individually. (*Q < 0.1; exact Q values are in the Source Data).

Source data

Extended Data Fig. 2 Mitochondrial complex III is required for T-ALL growth in vivo.

a, Schematic representation of the T-ALL experiments. b, c, Percentage of GFP+ T-ALL cells from the spleen (b) or bone marrow (c) of QPC-WT and QPC-KO recipients (WT: n =  7; KO: n = 5 mice). d, e, The absolute number of GFP+ T-ALL cells from the spleen (d) or bone marrow (e) of QPC-WT and QPC-KO recipients (WT: n =  7; KO: n =  5 mice). f, Weight of spleens from QPC-WT and QPC-KO recipients (WT: n =  6; KO: n =  4 mice). g, Survival of mice injected with QPC-WT or QPC-KO T-ALL cells (WT: n =  7; KO: n =  4 mice). Data are mean ± s.e.m. from three independent experiments. *P < 0.05, **P < 0.01, two-tailed t-tests with a Welch’s correction (exact P values are in the Source Data). Survival curves were compared using the log-rank test (P < 0.0001). An example of the gating strategy is provided in Supplementary Fig. 7.

Source data

Extended Data Fig. 3 Complex III-deficient cells are auxotrophic for uridine.

a, Schematic representation of the ETC in AOX expressing 143B-CYTB-Δ cells. b, Coupled OCR of 143B-CYTB-Δ-GFP and 143B-CYTB-Δ-AOX cells (n = 5 biologically independent experiments). c, Tumour mass of xenografts from 143B-CYTB-Δ-GFP and 143B-CYTB-Δ-AOX cells (n = 9 mice per group from two independent cohorts). d, Coupled OCR of KP-QPC_KO-GFP and KP-QPC_KO-AOX cells (n = 7 replicates from one representative of five biologically independent experiments). e, 143B-CYTB-WT treated or untreated with piericidin A (0.5 μM) or antimycin A (0.5 μM) were grown in the presence or absence of methyl pyruvate and/or uridine and cell number was assessed after 72 h (n = 4 biologically independent experiments). f, The dihydroorotate-to-orotate ratio was assessed in 143B-CYTB-WT treated or untreated with piericidin A (0.5 μM) or antimycin A (0.5 μM) (n = 6 biologically independent experiments). Data are mean ± s.e.m. (b, c, e, f) or mean ± s.d. (d). *P < 0.05, **P < 0.01, two-tailed t-tests (bd), two-way ANOVA (e) with a Bonferroni test for multiple comparisons or one-way ANOVA (f) with a Bonferroni test for multiple comparisons (exact P values are in the Source Data).

Source data

Extended Data Fig. 4 De novo pyrimidine synthesis is necessesary for tumour growth.

a, Schematic representation of the ETC in 143B-CYTB-Δ–DHODH_KO-AOX cells. b, Western blot analysis of DHODH in 143B-CYTB-Δ non-targeting (NT) and 143B-CYTB-Δ-DHODH_KO cells. Tubulin was used as a loading control. Data are representative of two independent experiments. c, 143B-CYTB-Δ-NT or 143B-CYTB-Δ-DHODH-KOs expressing GFP or AOX were grown in the presence or absence of uridine and cell number was assessed after 72 h (n = 5 biologically independent experiments). d, e, Average tumour volume (d) and tumour mass (e) of xenografts from 143B-CYTB-Δ-NT-AOX and 143B-CYTB-Δ-DHODH_KO2-AOX cells (n = 10 mice per group from two independent cohorts). f, Western blot analysis of DHODH protein levels in 143B-CYTB-Δ-NT, 143B-CYTB-Δ-DHODH_KO2-AOX-RFP and 143B-CYTB-Δ-DHODH_KO2-AOX-cDNA DHODH cells. Data are representative of three independent experiments. g, 143B-CYTB-Δ-DHODH_KO2-AOX-RFP and 143B-CYTB-Δ-DHODH_KO2-AOX-cDNA DHODH cells were grown in the presence or absence of uridine and cell number was assessed after 72 h (n = 5 biologically independent experiments). h, i, Average tumour volume (h) and tumour mass (i) of xenografts from 143B-CYTB-Δ-DHODH_KO2-AOX-RFP and 143B-CYTB-Δ-DHODH_KO2-AOX-cDNA DHODH cells (n = 9 mice per group from two independent cohorts). Data are mean ± s.e.m. (ce, gi) *P < 0.05, **P < 0.01, two-tailed t-tests (e, i) or two-way ANOVA (c, d, g, h) with a Bonferroni test for multiple comparisons (exact P values are in the Source Data). For gel source data, see Supplementary Fig. 4.

Source data

Extended Data Fig. 5 Restoration of complex I by ectopic expression of NDUFS2 cDNA rescues tumour growth.

a, Schematic representation of the ETC in complex I-deficient 143B-CYTB-Δ-NDUFS2_KO-AOX cells. b, Tumour mass of xenografts from 143B-CYTB-Δ-NT-AOX and 143B-CYTB-Δ-NDUFS2_KO1-AOX cells (n = 10 mice per group from two independent cohorts). c, Western blot analysis of NDUFS2 protein levels in 143B-CYTB-Δ-NT cells, and in AOX-expressing 143B-CYTB-Δ-NDUFS2_KO1 clone transduced with either RFP or human NDUFS2 cDNA. GAPDH was used as a loading control. Data representative of two independent experiments. d, Basal OCR of AOX expressing 143B-CYTB-Δ-NDUFS2_KO1 cells transduced with either RFP or human NDUFS2 cDNA. e,143B-CYTB-Δ-NDUFS2_KO1-AOX-RFP and 143B-CYTB-Δ-NDUFS2_KO1-AOX-cDNA NDUFS2 cells were grown in the presence or absence of methyl pyruvate and cell number was assessed after 72 h (n = 5 biologically independent experiments). f, g, Average tumour volume (f) and tumour mass (g) of xenografts from AOX-expressing 143B-CYTB-Δ-NDUFS2_KO1 cells transduced with either RFP or human NDUFS2 cDNA (n = 9 mice per group from two independent cohorts). Data are mean ± s.e.m. (b, dg). *P < 0.05, **P < 0.01, two-tailed t-tests (b, d, g) or two-way ANOVA (e, f) with a Bonferroni test for multiple comparisons (exact P values are in the Source Data). For gel source data, see Supplementary Fig. 2.

Source data

Extended Data Fig. 6 NDI1 expression in complex I-deficient cells rescues electron transfer but not ATP production.

a, Schematic representation of the ETC in complex I deficient 143B-CYTB-Δ-NDUFS2_KO-AOX cells expressing NDI1. b, 143B-CYTB-Δ-NDUFS2_KO1-AOX-RFP and 143B-CYTB-Δ-NDUFS2_KO1-AOX-NDI1 cells were grown in the presence or absence of methyl pyruvate and cell number was assessed after 72 h (n = 6 biologically independent experiments). c, The heat map displays the relative abundance of significantly changed metabolites in 143B-CYTB-Δ–NDUFS2_KO1-AOX cells expressing RFP, NDI1 or LbNOX in either mitochondria or cytosol (n = 4 biologically independent experiments). A red–blue colour scale depicts the abundance of the metabolites (red: high, blue: low). Metabolites levels were analysed with multiple one-way ANOVA using an FDR of 0.1 and Fisher’s least significant difference test post hoc analyses Q = 10%. Each row was analysed individually. (*Q < 0.1; exact Q values are in the Source Data.) d, 143B-CYTB-Δ-NT-AOX, 143B-CYTB-Δ-NDUFS2_KO1-AOX-RFP and 143B-CYTB-Δ-NDUFS2_KO1-AOX-NDI1 cells were grown in media containing 10 mM glucose or 10 mM galactose for 48 h and assessed for cell death (n = 4 biologically independent experiments). e, Tumour mass of xenografts from 143B-CYTB-Δ-NDUFS2_KO1 cells expressing AOX and either RFP or NDI1 (n = 10 mice per group from two independent cohorts). Data are mean ± s.e.m. (b, d, e). *P < 0.05, **P < 0.01, two-tailed t-tests (e) or two-way ANOVA (b, d) with a Bonferroni test for multiple comparisons (exact P values are in the Source Data).

Source data

Extended Data Fig. 7 LbNOX expression in mitochondria or cytosol promotes major changes in the metabolome of complex III-deficient cells.

a, Schematic representation of the ETC in 143B-CYTB-Δ cells expressing LbNOX in mitochondria. b, Intracellular NAD+/NADH ratio in 143B-CYTB-Δ-RFP, 143B-CYTB-Δ-LbNOX-Mito and 143B-CYTB-Δ-LbNOX-Cyto cells in the absence of methyl pyruvate (n = 5 biologically independent experiments). ce, Intracellular aspartate (c), succinate (d) and α-ketoglutarate levels (e) in 143B-CYTB-Δ-RFP, 143B-CYTB-Δ-LbNOX-Mito and 143B-CYTB-Δ-LbNOX-Cyto cells in the absence of methyl pyruvate (n = 5 biologically independent experiments). f, The heat map displays the relative abundance of significantly changed metabolites in 143B-CYTB-Δ-RFP, 143B-CYTB-Δ-LbNOX-Mito and 143B-CYTB-Δ-LbNOX-Cyto cells in the absence of methyl pyruvate (n = 5 biologically independent experiments). A red–blue colour scale depicts the abundance of the metabolites (red: high, blue: low). g, Tumour mass of xenografts from 143B-CYTB-Δ-RFP, 143B-CYTB-Δ-LbNOX-Mito and 143B-CYTB-Δ-LbNOX-Cyto cells (n = 9 mice per group from two independent cohorts). Data are mean ± s.e.m. (be, g). *P < 0.05, **P < 0.01, one-way ANOVA (b, g) with a Bonferroni test for multiple comparisons (exact P values are in the Source Data). Metabolites levels (cf) were analysed with multiple one-way ANOVA using an FDR of 0.1 and Fisher’s least significant difference test post hoc analyses Q = 10%. Each row was analysed individually (*Q < 0.1; exact Q values in Source Data).

Source data

Extended Data Fig. 8 LbNOX expression in mitochondria or cytosol promotes major changes in the metabolome of complex I deficient cells.

a, Schematic representation of the ETC in 143B-CYTB-Δ-NDUFS2_KO-AOX cells expressing LbNOX in mitochondria. b, 143B-CYTB-Δ-NDUFS2_KO1-AOX-LbNOX-Mito and 143B-CYTB-Δ-NDUFS2_KO1-AOX-LbNOX-Cyto were grown in the presence or absence of methyl pyruvate and cell number was assessed after 72 h (n = 5 biologically independent experiments). c, Intracellular NAD+/NADH ratio of 143B-CYTB-Δ-NDUFS2_KO1-AOX-RFP, 143B-CYTB-Δ-NDUFS2_KO1-AOX-LbNOX-Mito and 143B-CYTB-Δ-NDUFS2_KO1-AOX-LbNOX-Cyto cells in the absence of methyl pyruvate and uridine (n = 4 biologically independent experiments). d, Intracellular aspartate levels of 143B-CYTB-Δ-NDUFS2_KO1-AOX-RFP, 143B-CYTB-Δ-NDUFS2_KO1-AOX-LbNOX-Mito and 143B-CYTB-Δ-NDUFS2_KO1-AOX-LbNOX-Cyto cells in the absence of methyl pyruvate and uridine (n = 4 biologically independent experiments). e, 143B-CYTB-Δ-NDUFS2_KO1-AOX-LbNOX-Mito and 143B-CYTB-Δ-NDUFS2_KO1-AOX-LbNOX-Cyto cells were grown in medium containing 10 mM glucose or 10 mM galactose for 48 h and assessed for cell death (n = 4 biologically independent experiments). f, Tumour mass of xenografts from 143B-CYTB-Δ-NDUFS2_KO1-AOX cells expressing LbNOX in either mitochondria or cytosol (n = 10 mice per group from two independent cohorts). g, Western blot analysis (data representative of two independent experiments) of LbNOX expression in xenograft tumours from 143B-CYTB-Δ-NDUFS2_KO1-AOX-RFP, 143B-CYTB-Δ-NDUFS2_KO1-AOX-LbNOX-Mito and 143B-CYTB-Δ-NDUFS2_KO1-AOX-LbNOX-Cyto cells. Tubulin was used as a loading control. Data are mean ± s.e.m. (bf). *P < 0.05, **P < 0.01, two-tailed t-tests (f), one-way ANOVA (c) with a Bonferroni test for multiple comparisons or a two-way ANOVA (b, e) with a Bonferroni test for multiple comparisons (exact P values are in the Source Data). Metabolites levels (d) were analysed with multiple one-way ANOVA using an FDR of 0.1 and Fisher’s least significant difference test post hoc analyses Q = 10%. Each row was analysed individually. (*Q < 0.1; exact Q values in Source Data.) For gel source data, see Supplementary Fig. 5.

Source data

Extended Data Fig. 9 Complex I-deficient cells expressing LbNOX in the cytosol perform glutamine reductive carboxylation.

a, Schematic representation for oxidative and reductive glutamine metabolism. Metabolism of [U-13C]glutamine generates fully labelled α-ketoglutarate. Oxidation of α-ketoglutarate in the TCA cycle produces metabolites with four 13C-carbons (m + 4), while reduction of α-ketoglutarate through the reductive carboxylation pathway produces citrate with five 13C-carbons (m + 5). Further reductive metabolism of the m + 5 citrate yields metabolites with three 13C-carbons (m + 3). bh, 143B-CYTB-Δ-NDUFS2_KO1-AOX-RFP, 143B-CYTB-Δ-NDUFS2_KO1-AOX-LbNOX-Mito and 143B-CYTB-Δ-NDUFS2_KO1-AOX-LbNOX-Cyto cells were labelled for 6 h with [U-13C]glutamine in the presence (bd) or absence (eh) of methyl pyruvate, and the percentage of labelled metabolite pools was examined. m + 5 and m + 3 pools result from glutamine flow through reductive metabolism. m + 4 pools result from glutamine flow through oxidative metabolism. Data are mean ± s.e.m. of four biologically independent experiments.

Source data

Extended Data Fig. 10 Complex II is necessary for tumour growth.

a, Schematic representation of the ETC in complex II deficient 143B-CYTB-Δ cells expressing AOX. b, Western blot analysis of SDHA in 143B-CYTB-Δ non-targeting and 143B-CYTB-Δ-SDHA_KO cells. Tubulin was used as a loading control. Data representative of two independent experiments. c, Complex II-driven OCR of permeabilized 143B-CYTB-Δ-NT-AOX and 143B-CYTB-Δ-SDHA_KO2-AOX cells. Piericidin A (1 μM) and antimycin A (1 μM) were used to inhibit complex I and III, respectively. SHAM (2 mM) was used to inhibit AOX activity (n = 4 biologically independent experiments). d, 143B-CYTB-Δ-SDHA-KOs expressing GFP or AOX were grown in the presence or absence of methyl pyruvate and cell number was assessed after 72 h (n = 5 biologically independent experiments). e, f, Average tumour volume (e) and tumour mass (f) of xenografts from 143B-CYTB-Δ-NT-AOX and 143B-CYTB-Δ-SDHA_KO2-AOX cells (n = 8 mice per group from two independent cohorts). Data are mean ± s.e.m. (cf). *P < 0.05; **P < 0.01, two-tailed t-tests (f) or two-way ANOVA (d, e) with a Bonferroni test for multiple comparisons (exact P value are in the Source Data). For gel source data, see Supplementary Fig. 6.

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Extended Data Fig. 11 Restoration of complex II by ectopic expression of SDHA cDNA rescues tumour growth.

a, Western blot analysis of SDHA protein levels in 143B-CYTB-Δ-NT, 143B-CYTB-Δ-SDHA_KO2-AOX-RFP and 143B-CYTB-Δ-SDHA_KO2-AOX-cDNA SDHA cells. Data representative of three independent experiments. b, Complex II-driven OCR of permeabilized 143B-CYTB-Δ-SDHA_KO2-AOX-RFP and 143B-CYTB-Δ-SDHA_KO2-AOX-cDNA SDHA cells. Succinate and ADP were provided as substrates. Piericidin A (1 μM) and antimycin A (1 μM) were used to inhibit complex I and III respectively. SHAM (2 mM) was used to inhibit AOX activity (n = 4 biologically independent experiments). c, 143B-CYTB-Δ-SDHA_KO2-AOX-RFP and 143B-CYTB-Δ-SDHA_KO2-AOX-cDNA SDHA cells were grown in the presence or absence of methyl pyruvate and cell number was assessed after 72 h (n = 5 biologically independent experiments). d, e, Average tumour volume (d) and tumour mass (e) of xenografts from 143B-CYTB-Δ-SDHA_KO2-AOX-RFP and 143B-CYTB-Δ-SDHA_KO2-AOX-cDNA SDHA cells (n = 8 mice per group from two independent cohorts). Data are mean ± s.e.m. (be). *P < 0.05, **P < 0.01, two-tailed t-tests (e) or two-way ANOVA (c, d) with a Bonferroni test for multiple comparisons (exact P values are in the Source Data). For gel source data, see Supplementary Fig. 6.

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Supplementary Information

This file contains Supplementary Figures 1–6 (source data for western blots), Supplementary Figure 7 (the gating strategy for T-ALL experiments) and Supplementary Table 1 (the sequences of the sgRNAs used in this study).

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Martínez-Reyes, I., Cardona, L.R., Kong, H. et al. Mitochondrial ubiquinol oxidation is necessary for tumour growth. Nature 585, 288–292 (2020). https://doi.org/10.1038/s41586-020-2475-6

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