Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis

  1. Shintaro Watanuki
  2. Hiroshi Kobayashi  Is a corresponding author
  3. Yuki Sugiura  Is a corresponding author
  4. Masamichi Yamamoto
  5. Daiki Karigane
  6. Kohei Shiroshita
  7. Yuriko Sorimachi
  8. Shinya Fujita
  9. Takayuki Morikawa
  10. Shuhei Koide
  11. Motohiko Oshima
  12. Akira Nishiyama
  13. Koichi Murakami
  14. Miho Haraguchi
  15. Shinpei Tamaki
  16. Takehiro Yamamoto
  17. Tomohiro Yabushita
  18. Yosuke Tanaka
  19. Go Nagamatsu
  20. Hiroaki Honda
  21. Shinichiro Okamoto
  22. Nobuhito Goda
  23. Tomohiko Tamura
  24. Ayako Nakamura-Ishizu
  25. Makoto Suematsu
  26. Atsushi Iwama
  27. Toshio Suda
  28. Keiyo Takubo  Is a corresponding author
  1. Department of Stem Cell Biology, Research Institute, National Center for Global Health and Medicine, Japan
  2. Division of Hematology, Department of Medicine, Keio University School of Medicine, Japan
  3. Department of Cell Fate Biology and Stem Cell Medicine, Tohoku University Graduate School of Medicine, Japan
  4. Department of Biochemistry, Keio University School of Medicine, Japan
  5. Center for Cancer Immunotherapy and Immunobiology, Kyoto University Graduate School of Medicine, Japan
  6. Department of Research Promotion and Management, National Cerebral and Cardiovascular Center, Japan
  7. Department of Life Sciences and Medical BioScience, Waseda University School of Advanced Science and Engineering, Japan
  8. Division of Stem Cell and Molecular Medicine, Center for Stem Cell Biology and Regenerative Medicine, Japan
  9. Department of Immunology, Yokohama City University Graduate School of Medicine, Japan
  10. Advanced Medical Research Center, Yokohama City University, Japan
  11. Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Japan
  12. International Research Center for Medical Sciences, Kumamoto University, Japan
  13. Center for Advanced Assisted Reproductive Technologies, University of Yamanashi, Japan
  14. Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Japan
  15. Field of Human Disease Models, Major in Advanced Life Sciences and Medicine, Institute of Laboratory Animals, Japan
  16. Department of Microscopic and Developmental Anatomy, Tokyo Women's Medical University, Japan
  17. Live Imaging Center, Central Institute for Experimental Animals, Japan
  18. Cancer Science Institute of Singapore, National University of Singapore, Singapore
35 figures, 1 table and 7 additional files

Figures

Figure 1 with 3 supplements
HSC cell cycling increases overall glycolytic flux, but not flux into mitochondria.

(A) Experimental design used for glucose isotope tracer analysis in HSCs from 5-FU- or PBS-treated mice. (B) Heat map of metabolite levels in HSCs derived from mice treated with PBS or 5-FU. (C–F) …

Figure 1—figure supplement 1
Dependence on glycolysis increases with cell cycle progression of HSCs.

(A) Schematic illustration of 5-FU administration and analysis. (B) Representative staining plot of BM cells derived from mice 5 d (day 6) after treatment with PBS or 5-FU (day 1); note the gating …

Figure 1—figure supplement 2
antified metabolite pool in HSCs under quiescence, proliferation, or OXPHOS-inhibition.

(A) Comparison of metabolite levels in c-Kit enriched cells pre- and post-sorting. n=3 biological replicates for each group. ND: not detected. (B) Metabolic overview of U-13C6-glucose tracing among …

Figure 1—figure supplement 3
Mito Stress test results using the Seahorse flux analyzer.

(A–D) Overview of the Mito Stress test on the Seahorse flux analyzer for PBS- or 5-FU-treated HSCs (A) ECAR, (B) OCR and HSCs or MyPs (C) ECAR, (D) OCR.

OXPHOS inhibition activates compensatory glycolysis in HSCs.

(A) Experimental design used for glucose isotope tracer analysis in HSCs treated with the OXPHOS inhibitor oligomycin. (B) Heat map of metabolite levels detected by in vitro tracer analysis of U-13C6

Figure 3 with 2 supplements
Quantitative 13C-MFA of quiescent, proliferative, and stressed HSCs.

(A–C) Overview of quantitative 13C-MFA of PBS-treated HSCs (A), 5-FU-treated HSCs (B), and OXPHOS-inhibited HSCs (C). The representative net flux for each reaction with glucose uptake as 100 is …

Figure 3—figure supplement 1
Quantitative 13C-MFA of HSCs under quiescence, proliferation, and OXPHOS inhibition.

(A–C) Enzyme reaction flux values for each simulation (100 times in total) in PBS-treated (A), 5-FU-treated (B), and OXPHOS-inhibited HSCs (C). Flux values calculated in the same simulation are …

Figure 3—figure supplement 2
Quantified metabolite pool in HSCs from PBS- or 5-FU-treated mice.

(A) Experimental schema. (B–D) Heat maps of the glycolytic system (B), TCA cycle (C), PPP and NAS and glutathione labeling rates (D). (E–I) Labeling rates of Asp M+2 (E), Glu M+2 (F), IMP M+5 (G), …

Figure 4 with 2 supplements
PFKFB3 activates the glycolytic system in proliferating HSCs.

(A) Experimental design used to conduct real-time ATP analysis of HSCs treated with 5-FU or PBS. PLFA medium containing mitochondrial substrates (pyruvate, lactate, fatty acids, and amino acids) but …

Figure 4—figure supplement 1
Establishment of a real-time ATP concentration analysis system using GO-ATeam2.

(A) Representative plot of HSPC fractions from GO-ATeam2 +mice. The identified fractions are shown at the top of the graph and the upper gating of that fraction is shown in parentheses. (B–C) Number …

Figure 4—figure supplement 2
FAO is not active in proliferating HSCs.

(A–H) Results of real-time ATP analysis of PBS- (red) or 5-FU-treated (blue) HSCs (two-days (A–D) or 14 days (E–H) after PBS/5-FU administration) after treatment with 2-DG (A, C, E, G) or oligomycin …

Figure 5 with 1 supplement
PFKFB3 accelerates glycolysis in HSCs under OXPHOS inhibition in an AMPK-dependent manner.

(A) Experimental design of real-time ATP analysis using GO-ATeam2 knock-in BMMNCs. Ba-M was used in experiments with oligomycin. For other experiments, PLFA medium was used. (B–C) Evaluation of …

Figure 5—figure supplement 1
Steady-state PFKFB3 activity defines HSC and HPC metabolic kinetics and cell cycle.

(A–D) Evaluation of factors affecting ATP concentration in MPPs (A), MEPs (B), CMPs (C), and CLPs (D) based on the GO-ATeam2 system. GO-ATeam2-knock-in BMMNCs were incubated with glucose, …

Figure 6 with 1 supplement
PFKFB3 methylation by PRMT1 enables ATP production by cell-cycling HSCs.

(A) Normalized Pfkfb3 mRNA counts based on RNA sequencing of PBS-treated (red) or 5-FU-treated (blue) HSCs. Data are representative results of pooled samples from three biological replicates. Data …

Figure 6—figure supplement 1
ene expression related to Prmt1 in proliferating HSCs.

Data are presented as the mean ± SD. * p≤0.05, ** p≤0.01, *** p≤0.001 as determined using a Student’s t-test.

Figure 7 with 1 supplement
PFKFB3 maintains HSC function under proliferative stress.

(A–C) Transplant analysis of Pfkfb3-KO or Pfkfb3CA-overexpressing HSCs. Experimental design (A). PB chimerism of donor-derived cells at 4 months post-transplant. Pfkfb3-KO group, n=6; Rosa26-KO …

Figure 7—figure supplement 1
PFKFB3 contributes to HSC proliferation and differentiation in vitro.

(A–D) Effects of in vitro PFKFB3 inhibition, KO, or overexpression on HSCs. Experimental design (A). Number of cells in an HSC-derived colony following exposure to a PFKFB3 inhibitor (PFKFB3i) at …

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Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus, male and female)C57BL/6JJmsSlc Ly5.2+Japan SLC (Shizuoka, Japan)N/A
Strain, strain background (Mus musculus, male and female)C57BL/6J-Ly5.1CLEA Japan (Shizuoka, Japan)N/AUtilized for hematopoietic cell transplantation studies to distinguish donor and recipient cells.
Strain, strain background (Mus musculus, male and female)GO-ATeam2 miceGenerated in Yamamoto laboratoryN/AUsed for ATP analysis
Strain, strain background (Mus musculus, male and female)Ubc-GFP miceThe Jackson LaboratoryStock No: 007076
Strain, strain background (Mus musculus, male and female)mVenus-p27K- miceProvided by Kitamura LaboratoryN/AUsed for cell cycle analysis
AntibodyAnti-mouse CD4-PerCP-Cy5.5
(clone: RM4-5, rat monoclonal)
TONBO biosciencesCat# 65–0042 U100; RRID: AB_2621876(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse CD8a-PerCP-Cy5.5
(clone: 53–6.7, rat monoclonal)
TONBO biosciencesCat# 65–0081 U100; RRID: AB_2621882(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse B220-PerCP-Cy5.5
(clone: RA3-6B2, rat monoclonal)
TONBO biosciencesCat# 65–0452 U100; RRID: AB_2621892(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse B220-APC
(clone: RA3-6B2, rat monoclonal)
BioLegendCat# 103212; RRID: AB_312997(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse Ter-119-PerCP-Cy5.5
(clone: TER-119, rat monoclonal)
TONBO biosciencesCat# 65–5921 U100(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse Gr1 (Ly-6G/6 C)-PerCP-Cy5.5
(clone: RB6-8C5, rat monoclonal)
BioLegendCat# 108428; RRID: AB_893558(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse Gr1-PE-Cy7
(clone: RB6-8C5, rat monoclonal)
TONBO biosciencesCat# 60–5931 U100; RRID: AB_2621870(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse Mac1 (CD11b)-PerCP-Cy5.5
(clone: M1/70, rat monoclonal)
TONBO biosciencesCat# 65–0112 U100; RRID: AB_2621885(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse Mac1-PE-Cy7
(clone: M1/70, rat monoclonal)
TONBO biosciencesCat# 60–0112 U100; RRID: AB_2621836(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse CD45.1-PE
(clone: A20, mouse monoclonal)
BD biosciencesCat# 553776; RRID: AB_395044(1 µL/mouse)
AntibodyAnti-mouse CD45.1-Alexa Fluor700
(clone: A20, mouse monoclonal)
BioLegendCat# 110724; RRID: AB_493733(1 µL/mouse)
AntibodyAnti-mouse CD45.2-FITC
(clone: 104, mouse monoclonal)
BD biosciencesCat# 553772; RRID: AB_395041(1 µL/mouse)
AntibodyAnti-mouse Sca-1 (Ly-6A/E)-PE-Cy7
(clone: E13-161.7, rat monoclonal)
BioLegendCat# 122514; RRID: AB_756199(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse c-Kit (CD117)-APC-Cy7
(clone: 2B8, rat monoclonal)
BioLegendCat# 105826; RRID: AB_1626278(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyCD117 MicroBeads MouseMiltenyi BiotecCat# 130-091-224(1:5)
AntibodyAnti-mouse CD150-PE
(clone: TC15-12F12.2, rat monoclonal)
BioLegendCat# 115904; RRID: AB_313683(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse CD150-BV421
(clone: TC15-12F12.2, rat monoclonal)
BioLegendCat# 115926; RRID: AB_2562190(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse CD150-APC
(clone: TC15-12F12.2, armenian hamster monoclonal)
BioLegendCat# 115910; RRID: AB_ 493460(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse CD48-FITC
(clone: HM48-1, armenian hamster monoclonal)
BioLegendCat# 103404; RRID: AB_313019(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse CD48-APC
(clone: HM48-1, qrmenian hamster monoclonal)
BioLegendCat# 103411; RRID: AB_571996(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse CD48-BV510
(clone: HM48-1, armenian hamster monoclonal)
BD biosciencesCat# 563536(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse CD48-Alexa Fluor700
(clone: HM48-1, armenian hamster monoclonal)
BioLegendCat# 103426
RRID: AB_10612754
(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-mouse CD41-APC
(clone: MWReg30, rat monoclonal)
BioLegendCat# 133914; RRID: AB_11125581(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-CD34-BV421
(clone: RAM34, rat monoclonal)
BD biosciencesCat# 562608; RRID: AB_11154576(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-CD34-FITC
(clone: RAM34, rat monoclonal)
InvitrogenCat# 11-0341-82; RRID: AB_465021(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-Flt3 (CD135)-APC
(clone: A2F10, rat monoclonal)
BioLegendCat# 135310; RRID: AB_2107050(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-CD127 (IL-7Rα)
(clone: A7R34, rat monoclonal)
BioLegendCat# 135023; RRID: AB_10897948(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-CD201 (EPCR)-PE (clone: RCR-16, rat monoclonal)BioLegendCat# 141504; RRID: AB_10899579(0.5 µL, 1 µL, or 2 µL/mouse)
AntibodyAnti-Ki67-Alexa Fluor555
(clone: B56, mouse monoclonal)
BD biosciencesCat# 558617(10 µL/sample)
AntibodyAnti-Ki67 Monoclonal Antibody (SolA15), eFluor 660, eBioscience
(Clone: SolA15, mouse monoclonal)
InvitrogenCat# 50-5698-82; RRID: AB_2574235(10 µL/sample)
AntibodyFc-block (anti-mouse CD16/32)
(clone: 2.4-G2, rat monoclonal)
BD biosciencesCat# 553142; RRID: AB_394657(2 µL/mouse)
AntibodyAnti-CD16/CD32 Monoclonal Antibody (93),
Alexa Fluor 700 (clone: 93, rat monoclonal)
InvitrogenCat# 56-0161-82; RRID: AB_493994(2 µL/mouse)
AntibodyPhospho-Rb (Ser807/811) (D20B12) XP (rabbit monoclonal)Cell Signaling TechnologyCat# 8516(1:200)
AntibodyAnti-human CD8-APC (clone: SK1, mouse monoclonal)BiolegendCat# 344721; RRID: AB_2075390(1 µL/sample)
AntibodyRecombinant anti-PFKFB3 antibody (rabbit monoclonal)AbcamCat# ab181861(1:100)
AntibodyAnti-PFK2 (Ser467) antibody (rabbit polyclonal)BiossCat# bs-3331R(1:100)
AntibodyRecombinant anti-methyl-PFKFB3 antibody (rabbit polyclonal)Obtained from
Takehiro Yamamoto at Keio University
DOI: 10.1038/ncomms4480
N/A(1:100)
Gene (Mus musculus)Pfkfb3This paperN/ADetails are as described in Methods
Gene (Mus musculus)Pfkfb3CAThis paperN/ADetails are as described in Methods
Recombinant DNA reagentpMYs-IRES-GFPObtained from
Toshio Kitamura at IMUST
N/AUsed as backbone vector for gene overexpression
Recombinant DNA reagentpMYs-IRES-human CD8Obtained from
Go Nagamatsu at Kyushu University
N/AUsed as backbone vector for gene overexpression
Recombinant DNA reagentPfkfb3-knockout gRNA (s)Custom made in lab or purchased from Synthego, Inc.N/ADetails are as described in Methods
Recombinant DNA reagentRosa-knockout gRNACustom made in labN/ADetails are as described in Methods
Recombinant DNA reagentCD45-knockout gRNACustom made in labN/ADetails are as described in Methods
Chemical compound, drugISTThermo Fisher ScientificCat# 41400–045
Chemical compound, drugPenicilinMeiji SeikaPGLD755
Chemical compound, drugStreptomycin sulfateMeiji SeikaSSDN1013
Chemical compound, drugSodium seleniteNacalai TesqueCat# 11707–04
Chemical compound, drugFetal bovine serumBiowestCat# S1820-500
Chemical compound, drugFetal bovine serumThermo Fisher ScientificCat# 10270–106
Chemical compound, drugBovine serum albuminSigma AldrichCat# A4503-50G/100 G
Chemical compound, drug2-mercapto ethanol (2-ME) 1000 xLife TechnologiesCat# 21985–023
Chemical compound, drugThymidineTokyo Chemical Industry Co., Ltd.Cat# T0233
Chemical compound, drugRPMI 1640 Amino Acids Solution (50×)Sigma AldrichCat# R7131
Chemical compound, drugMEM Vitamin Solution (100×)Sigma AldrichCat# M6895
Chemical compound, drugL-glutamineSigma AldrichCat# G8540
Chemical compound, drugL-alanineSigma AldrichCat# A7469
Chemical compound, drugL-SerineSigma AldrichCat# S4311
Chemical compound, drugD(+)-GlucoseWakoCat# 049–31165
Chemical compound, drug13C-glucoseSigma AldrichCat# 389374
Chemical compound, drug2-NBDGCayman ChemicalCat# 11046
Chemical compound, drugCytochalasin BWakoCat# 030–17551
Chemical compound, drugPhloretinTCI chemicalsCat# P1966
Chemical compound, drug2-morpholinoethanesulfonic acidWakoCat# 341–01622
Chemical compound, drugmethionine sulfoneAlfa AesarCat# A17027
Chemical compound, drugSodium L-lactateSigma AldrichCat# L7022
Chemical compound, drugCholesterol Lipid Concentrate (250 X)GibcoCat# 12531018
Chemical compound, drug100mM-Sodium Pyruvate SolutionNacalai tesqueCat# 06977–34
Chemical compound, drugSodium HydroxideWakoCat# 194–18865
Chemical compound, drug5-fluorouracilKyowa Hakko KirinN/A
Chemical compound, drug2-Deoxy-D-GlucoseTokyo Chemical Industry Co., Ltd.Cat# D0051
Chemical compound, drugOligomycinCell Signaling TechnologyCat# 9996 L
Chemical compound, drugFCCPSigma AldrichCat# C2920
Chemical compound, drugRotenoneSigma AldrichCat# R8875
Chemical compound, drugEtomoxir (sodium salt)Cayman chemicalCat# 11969
Chemical compound, drug6-diazo-5-oxo-L-nor-LeucineCayman chemicalCat# 17580
Chemical compound, drugVerapamilSigma AldrichCat# V4629
Chemical compound, drugN-acetyl-cysteineTokyo Chemical Industry Co., Ltd.Cat# A0905
Chemical compound, drugAZ PFKFB3 26R&D systemsCat# 5675
Chemical compound, drugDorsomorphin dihydrochlorideSanta Cruz BiotechnologyCat# sc-361173
Chemical compound, drugLKB1/AAK1 dual inhibitorChem SceneCat# CS-0342
Chemical compound, drugPKM2 inhibitor(compound 3 k)SelleckCat# S8616
Chemical compound, drugRecombinant Murine SCFPeproTechCat# 250–03
Chemical compound, drugRecombinant Human TPOPeproTechCat# 300–18
Chemical compound, drugα-hemolysinSigma AldrichCat# H9395
Chemical compound, drugPotassium ChlorideWakoCat# 7447-40-7
Chemical compound, drugSodium ChlorideWakoCat# 7647-14-5
Chemical compound, drugCalcium Nitrate TetrahydrateWakoCat# 13477-34-4
Chemical compound, drugMagnesium Sulfate (Anhydrous)WakoCat# 7487-88-9
Chemical compound, drugSodium Hydrogen CarbonateWakoCat# 144-55-8
Chemical compound, drugDisodium Hydrogenphosphate 12-WaterWakoCat# 10039-32-4
Chemical compound, drugGlutathione reduced formTokyo Chemical Industry Co., Ltd.Cat# G0074
Chemical compound, drugEthylene Glycol Bis(β-aminoethylether)-N,N,N',N'-tetraacetic AcidNacalai tesqueCat# 15214–21
Chemical compound, drugHEPESWakoCat# 7365-45-9
Chemical compound, drugMagnesium ChlorideWakoCat# 7786-30-3
Chemical compound, drugAdenosine 5’-triphosphate magnesium saltSigma AldrichCat# A9187
Chemical compound, drugDMSOSigma AldrichCat# D8418
Chemical compound, drugEthanolNacalai tesqueCat# 14712–63
Chemical compound, drugMethanolNacalai tesqueCat# 21914–03
Chemical compound, drugChloroformNacalai tesqueCat# 08401–65
Chemical compound, drugHoechst 33432Thermo Fisher ScientificCat# H3570(10 µg/mL)
Chemical compound, drugPropidium iodideThermo Fisher ScientificCat# P3566(1:1000)
Chemical compound, drugFlow-Check FluorspheresBeckman CoulterCat# 7547053
Chemical compound, drugTrueCut Cas9 Protein v2Thermo Fisher ScientificCat# A36498
Chemical compound, drugExTaqTakara bioCat# RR001
Chemical compound, drugNotINippon GeneCat# 312–01453
Chemical compound, drugEcoRINippon GeneCat# 314–00112
Chemical compound, drugRetroNectin (Recombinant Human Fibronectin Fragment)TakaraCat# T100A
Chemical compound, drugUltraPure DNase_RNase-Free Distilled WaterInvitrogenCat# 10977015
Chemical compound, drugGSK3368715MedChemExpressCat# HY-128717A
Commercial assay or kitRNeasy Mini KitQIAGENCat# 74104
Commercial assay or kitSuperScript VILOThermo Fisher ScientificCat# 11754–050
Commercial assay or kit2-mercapto ethanolSigma AldrichCat# M6250
Commercial assay or kitFlow Cytometry Size Calibration Kit (nonfluorescent microspheres)InvitrogenCat# F13838
Commercial assay or kit‘’Cellno’’ ATP assay reagent Ver.2Toyo B-Net CorporationCA2-50
Commercial assay or kitFixation and Permeabilization SolutionBD BiosciencesCat# 554722
Commercial assay or kitPerm/Wash BufferBD BiosciencesCat# 554723
Commercial assay or kitCellROX Deep Red ReagentInvitrogenCat# C10422
Commercial assay or kitSMART-Seq v4 Ultra Low Input RNA Kit for SequencingClontechCat# Z4888N
Commercial assay or kitNEBNext Ultra DNA Library Prep Kit for IlluminaNew England BioLabsCat# E7370S
Commercial assay or kitCUGA7 gRNA Synthesis KitNippon GeneCat# 314–08691
Commercial assay or kitExtract-N-Amp Blood PCR KitMerckCat# XNAB2-1KT
Commercial assay or kitWizard SV Gel and PCR Clean-Up SystemPromegaCat# A9281
Commercial assay or kitBD Pharmingen FITC BrdU Flow KitBD BiosciencesCat# 559619
Commercial assay or kitBD Pharmingen PE Annexin V Apoptosis Detection Kit IBD BiosciencesCat# 559763
Commercial assay or kitFAOBlueFunakoshiCat# FDV-0033
Software, algorithmR v3.5.2R Development Core Team, 2018http://www.r-project.org
Software, algorithmTopHat v2.0.1310.1186/gb-2013-14-4-r36; Kim et al., 2013https://ccb.jhu.edu/software/tophat/index.shtml
Software, algorithmCufflinks v2.2.110.1038/nbt.1621; Trapnell et al., 2012http://cole-trapnell-lab.github.io/cufflinks/
Software, algorithmGSEA software v4.3.0Broad Institute; Subramanian et al., 2005https://www.gsea-msigdb.org/gsea/index.jsp
Software, algorithmFlowJo version 9BD Bioscienceshttps://www.flowjo.com/
Software, algorithmTIDE v3.3.0DOI:10.1093/nar/gku93; Brinkman et al., 2014https://tide.nki.nl/
Software, algorithmOpenMebiusDOI:10.1155/2014/627014; Kajihata et al., 2014http://www-shimizu.ist.osaka-u.ac.jp/hp/en/software/OpenMebius.html

Additional files

Supplementary file 1

Custom RPMI medium for culture and ATP analysis.

Composition of custom RPMI medium for culture (upper) and ATP analysis (lower). “-“ means 0 mg/L.

https://cdn.elifesciences.org/articles/87674/elife-87674-supp1-v1.xlsx
Supplementary file 2

In vitro tracer analysis for 5-FU-treated HSCs.

Results of tracer analysis using U-13C6-glucose with HSCs from mice treated with PBS or 5-FU. Each section contains raw data from the glycolytic system, TCA cycle, and P~NAS from top to bottom. Data from three individual experiments are described for each. All values represent average metabolite levels in single HSCs obtained by dividing the metabolite levels detected in HSCs (compared to internal standards) by the number of HSCs used in the analysis.

https://cdn.elifesciences.org/articles/87674/elife-87674-supp2-v1.xlsx
Supplementary file 3

In vitro tracer analysis for oligomycin-treated HSCs.

Results of tracer analysis using U-13C6-glucose with HSCs treated with DMSO (Oligomycin-) or oligomycin (Oligomycin+). Each section contains raw data from the glycolytic system, TCA cycle, and P~NAS from top to bottom. Data from four individual experiments are described for each. All values represent average metabolite levels in single HSCs, obtained by dividing the metabolite levels detected in HSCs (compared to internal standards) by the number of HSCs used in the analysis.

https://cdn.elifesciences.org/articles/87674/elife-87674-supp3-v1.xlsx
Supplementary file 4

13C quantitative metabolic flux analysis.

Metabolic flux values of each enzyme obtained from 100 trials of 13C quantitative metabolic flux analysis for PBS-treated (left), 5-FU-treated (middle), and OXPHOS-inhibited HSCs (right).

https://cdn.elifesciences.org/articles/87674/elife-87674-supp4-v1.xlsx
Supplementary file 5

In vivo tracer analysis for 5-FU treated mice.

Results of tracer analysis during continuous in vivo administration of U-13C6-glucose to mice treated with 5-FU or PBS. A sheet is prepared for each metabolite and each contains two tables. The A.U. table (left) shows the metabolite levels detected in the four biological replicates in the 5-FU and PBS groups, obtained by dividing the metabolite levels detected in HSCs (compared to internal standards) by the number of HSCs used in the analysis. The ratio table (right) shows the calculated percentage of labeled metabolites among detected metabolites, where 12 C indicates unlabeled metabolites and 13Cn indicates n-carbon labeled metabolites by U-13C6-glucose.

https://cdn.elifesciences.org/articles/87674/elife-87674-supp5-v1.xlsx
Supplementary file 6

Primer list for genotyping PCR.

https://cdn.elifesciences.org/articles/87674/elife-87674-supp6-v1.xlsx
MDAR checklist
https://cdn.elifesciences.org/articles/87674/elife-87674-mdarchecklist1-v1.docx

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