Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis
- Department of Stem Cell Biology, Research Institute, National Center for Global Health and Medicine, Japan
- Division of Hematology, Department of Medicine, Keio University School of Medicine, Japan
- Department of Cell Fate Biology and Stem Cell Medicine, Tohoku University Graduate School of Medicine, Japan
- Department of Biochemistry, Keio University School of Medicine, Japan
- Center for Cancer Immunotherapy and Immunobiology, Kyoto University Graduate School of Medicine, Japan
- Department of Research Promotion and Management, National Cerebral and Cardiovascular Center, Japan
- Department of Life Sciences and Medical BioScience, Waseda University School of Advanced Science and Engineering, Japan
- Division of Stem Cell and Molecular Medicine, Center for Stem Cell Biology and Regenerative Medicine, Japan
- Department of Immunology, Yokohama City University Graduate School of Medicine, Japan
- Advanced Medical Research Center, Yokohama City University, Japan
- Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Japan
- International Research Center for Medical Sciences, Kumamoto University, Japan
- Center for Advanced Assisted Reproductive Technologies, University of Yamanashi, Japan
- Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Japan
- Field of Human Disease Models, Major in Advanced Life Sciences and Medicine, Institute of Laboratory Animals, Japan
- Department of Microscopic and Developmental Anatomy, Tokyo Women's Medical University, Japan
- Live Imaging Center, Central Institute for Experimental Animals, Japan
- Cancer Science Institute of Singapore, National University of Singapore, Singapore
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) …
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Figure 1—source data 1
Raw data for Figure 1B–H, K and L.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig1-data1-v1.xlsx
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 …
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Figure 1—figure supplement 1—source data 1
Raw data for Figure 1—figure supplement 1D–F, H, K-N.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig1-figsupp1-data1-v1.xlsx
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 …
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Figure 1—figure supplement 2—source data 1
Raw data for Figure 1—figure supplement 2A–H.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig1-figsupp2-data1-v1.xlsx
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.
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Figure 1—figure supplement 3—source data 1
Raw data for Figure 1—figure supplement 3A–D.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig1-figsupp3-data1-v1.xlsx
Figure 2
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…
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Figure 2—source data 1
Raw data for Figure 2B–H.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig2-data1-v1.xlsx
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 …
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Figure 3—source data 1
Raw data for Figure 3D–L.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig3-data1-v1.xlsx
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 …
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Figure 3—figure supplement 1—source data 1
Raw data for Figure 3—figure supplement 1D–U.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig3-figsupp1-data1-v1.xlsx
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), …
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Figure 3—figure supplement 2—source data 1
Raw data for Figure 3—figure supplement 2B–J.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig3-figsupp2-data1-v1.xlsx
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 …
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Figure 4—source data 1
Raw data for Figure 4D–F, I and J.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig4-data1-v1.xlsx
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 …
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Figure 4—figure supplement 1—source data 1
Raw data for Figure 4—figure supplement 1B, C and I.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig4-figsupp1-data1-v1.xlsx
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 …
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Figure 4—figure supplement 2—source data 1
Raw data for Figure 4—figure supplement 2C, D, G-I, K, M, N and P.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig4-figsupp2-data1-v1.xlsx
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 …
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Figure 5—source data 1
Raw data for Figure 5D, E, H, I, K, L and M.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig5-data1-v1.xlsx
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, …
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Figure 5—figure supplement 1—source data 1
Raw data for Figure 5—figure supplement 1F,J-L, Q.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig5-figsupp1-data1-v1.xlsx
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 …
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Figure 6—source data 1
Raw data for Figure 6A–J.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig6-data1-v1.xlsx
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.
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Figure 6—figure supplement 1—source data 1
Raw data for Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig6-figsupp1-data1-v1.xlsx
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 …
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Figure 7—source data 1
Raw data for Figure 7B, C, E, I–K and N.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig7-data1-v1.xlsx
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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Figure 7—figure supplement 1—source data 1
Raw data for Figure 7—figure supplement 1B–D, G, H.
- https://cdn.elifesciences.org/articles/87674/elife-87674-fig7-figsupp1-data1-v1.xlsx
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Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional 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-Ly5.1 | CLEA Japan (Shizuoka, Japan) | N/A | Utilized for hematopoietic cell transplantation studies to distinguish donor and recipient cells. |
| Strain, strain background (Mus musculus, male and female) | GO-ATeam2 mice | Generated in Yamamoto laboratory | N/A | Used for ATP analysis |
| Strain, strain background (Mus musculus, male and female) | Ubc-GFP mice | The Jackson Laboratory | Stock No: 007076 | |
| Strain, strain background (Mus musculus, male and female) | mVenus-p27K- mice | Provided by Kitamura Laboratory | N/A | Used for cell cycle analysis |
| Antibody | Anti-mouse CD4-PerCP-Cy5.5 (clone: RM4-5, rat monoclonal) | TONBO biosciences | Cat# 65–0042 U100; RRID: AB_2621876 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse CD8a-PerCP-Cy5.5 (clone: 53–6.7, rat monoclonal) | TONBO biosciences | Cat# 65–0081 U100; RRID: AB_2621882 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse B220-PerCP-Cy5.5 (clone: RA3-6B2, rat monoclonal) | TONBO biosciences | Cat# 65–0452 U100; RRID: AB_2621892 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse B220-APC (clone: RA3-6B2, rat monoclonal) | BioLegend | Cat# 103212; RRID: AB_312997 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse Ter-119-PerCP-Cy5.5 (clone: TER-119, rat monoclonal) | TONBO biosciences | Cat# 65–5921 U100 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse Gr1 (Ly-6G/6 C)-PerCP-Cy5.5 (clone: RB6-8C5, rat monoclonal) | BioLegend | Cat# 108428; RRID: AB_893558 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse Gr1-PE-Cy7 (clone: RB6-8C5, rat monoclonal) | TONBO biosciences | Cat# 60–5931 U100; RRID: AB_2621870 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse Mac1 (CD11b)-PerCP-Cy5.5 (clone: M1/70, rat monoclonal) | TONBO biosciences | Cat# 65–0112 U100; RRID: AB_2621885 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse Mac1-PE-Cy7 (clone: M1/70, rat monoclonal) | TONBO biosciences | Cat# 60–0112 U100; RRID: AB_2621836 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse CD45.1-PE (clone: A20, mouse monoclonal) | BD biosciences | Cat# 553776; RRID: AB_395044 | (1 µL/mouse) |
| Antibody | Anti-mouse CD45.1-Alexa Fluor700 (clone: A20, mouse monoclonal) | BioLegend | Cat# 110724; RRID: AB_493733 | (1 µL/mouse) |
| Antibody | Anti-mouse CD45.2-FITC (clone: 104, mouse monoclonal) | BD biosciences | Cat# 553772; RRID: AB_395041 | (1 µL/mouse) |
| Antibody | Anti-mouse Sca-1 (Ly-6A/E)-PE-Cy7 (clone: E13-161.7, rat monoclonal) | BioLegend | Cat# 122514; RRID: AB_756199 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse c-Kit (CD117)-APC-Cy7 (clone: 2B8, rat monoclonal) | BioLegend | Cat# 105826; RRID: AB_1626278 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | CD117 MicroBeads Mouse | Miltenyi Biotec | Cat# 130-091-224 | (1:5) |
| Antibody | Anti-mouse CD150-PE (clone: TC15-12F12.2, rat monoclonal) | BioLegend | Cat# 115904; RRID: AB_313683 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse CD150-BV421 (clone: TC15-12F12.2, rat monoclonal) | BioLegend | Cat# 115926; RRID: AB_2562190 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse CD150-APC (clone: TC15-12F12.2, armenian hamster monoclonal) | BioLegend | Cat# 115910; RRID: AB_ 493460 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse CD48-FITC (clone: HM48-1, armenian hamster monoclonal) | BioLegend | Cat# 103404; RRID: AB_313019 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse CD48-APC (clone: HM48-1, qrmenian hamster monoclonal) | BioLegend | Cat# 103411; RRID: AB_571996 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse CD48-BV510 (clone: HM48-1, armenian hamster monoclonal) | BD biosciences | Cat# 563536 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse CD48-Alexa Fluor700 (clone: HM48-1, armenian hamster monoclonal) | BioLegend | Cat# 103426 RRID: AB_10612754 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-mouse CD41-APC (clone: MWReg30, rat monoclonal) | BioLegend | Cat# 133914; RRID: AB_11125581 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-CD34-BV421 (clone: RAM34, rat monoclonal) | BD biosciences | Cat# 562608; RRID: AB_11154576 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-CD34-FITC (clone: RAM34, rat monoclonal) | Invitrogen | Cat# 11-0341-82; RRID: AB_465021 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-Flt3 (CD135)-APC (clone: A2F10, rat monoclonal) | BioLegend | Cat# 135310; RRID: AB_2107050 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-CD127 (IL-7Rα) (clone: A7R34, rat monoclonal) | BioLegend | Cat# 135023; RRID: AB_10897948 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-CD201 (EPCR)-PE (clone: RCR-16, rat monoclonal) | BioLegend | Cat# 141504; RRID: AB_10899579 | (0.5 µL, 1 µL, or 2 µL/mouse) |
| Antibody | Anti-Ki67-Alexa Fluor555 (clone: B56, mouse monoclonal) | BD biosciences | Cat# 558617 | (10 µL/sample) |
| Antibody | Anti-Ki67 Monoclonal Antibody (SolA15), eFluor 660, eBioscience (Clone: SolA15, mouse monoclonal) | Invitrogen | Cat# 50-5698-82; RRID: AB_2574235 | (10 µL/sample) |
| Antibody | Fc-block (anti-mouse CD16/32) (clone: 2.4-G2, rat monoclonal) | BD biosciences | Cat# 553142; RRID: AB_394657 | (2 µL/mouse) |
| Antibody | Anti-CD16/CD32 Monoclonal Antibody (93), Alexa Fluor 700 (clone: 93, rat monoclonal) | Invitrogen | Cat# 56-0161-82; RRID: AB_493994 | (2 µL/mouse) |
| Antibody | Phospho-Rb (Ser807/811) (D20B12) XP (rabbit monoclonal) | Cell Signaling Technology | Cat# 8516 | (1:200) |
| Antibody | Anti-human CD8-APC (clone: SK1, mouse monoclonal) | Biolegend | Cat# 344721; RRID: AB_2075390 | (1 µL/sample) |
| Antibody | Recombinant anti-PFKFB3 antibody (rabbit monoclonal) | Abcam | Cat# ab181861 | (1:100) |
| Antibody | Anti-PFK2 (Ser467) antibody (rabbit polyclonal) | Bioss | Cat# bs-3331R | (1:100) |
| Antibody | Recombinant anti-methyl-PFKFB3 antibody (rabbit polyclonal) | Obtained from Takehiro Yamamoto at Keio University DOI: 10.1038/ncomms4480 | N/A | (1:100) |
| Gene (Mus musculus) | Pfkfb3 | This paper | N/A | Details are as described in Methods |
| Gene (Mus musculus) | Pfkfb3CA | This paper | N/A | Details are as described in Methods |
| Recombinant DNA reagent | pMYs-IRES-GFP | Obtained from Toshio Kitamura at IMUST | N/A | Used as backbone vector for gene overexpression |
| Recombinant DNA reagent | pMYs-IRES-human CD8 | Obtained from Go Nagamatsu at Kyushu University | N/A | Used as backbone vector for gene overexpression |
| Recombinant DNA reagent | Pfkfb3-knockout gRNA (s) | Custom made in lab or purchased from Synthego, Inc. | N/A | Details are as described in Methods |
| Recombinant DNA reagent | Rosa-knockout gRNA | Custom made in lab | N/A | Details are as described in Methods |
| Recombinant DNA reagent | CD45-knockout gRNA | Custom made in lab | N/A | Details are as described in Methods |
| Chemical compound, drug | IST | Thermo Fisher Scientific | Cat# 41400–045 | |
| Chemical compound, drug | Penicilin | Meiji Seika | PGLD755 | |
| Chemical compound, drug | Streptomycin sulfate | Meiji Seika | SSDN1013 | |
| Chemical compound, drug | Sodium selenite | Nacalai Tesque | Cat# 11707–04 | |
| Chemical compound, drug | Fetal bovine serum | Biowest | Cat# S1820-500 | |
| Chemical compound, drug | Fetal bovine serum | Thermo Fisher Scientific | Cat# 10270–106 | |
| Chemical compound, drug | Bovine serum albumin | Sigma Aldrich | Cat# A4503-50G/100 G | |
| Chemical compound, drug | 2-mercapto ethanol (2-ME) 1000 x | Life Technologies | Cat# 21985–023 | |
| Chemical compound, drug | Thymidine | Tokyo Chemical Industry Co., Ltd. | Cat# T0233 | |
| Chemical compound, drug | RPMI 1640 Amino Acids Solution (50×) | Sigma Aldrich | Cat# R7131 | |
| Chemical compound, drug | MEM Vitamin Solution (100×) | Sigma Aldrich | Cat# M6895 | |
| Chemical compound, drug | L-glutamine | Sigma Aldrich | Cat# G8540 | |
| Chemical compound, drug | L-alanine | Sigma Aldrich | Cat# A7469 | |
| Chemical compound, drug | L-Serine | Sigma Aldrich | Cat# S4311 | |
| Chemical compound, drug | D(+)-Glucose | Wako | Cat# 049–31165 | |
| Chemical compound, drug | 13C-glucose | Sigma Aldrich | Cat# 389374 | |
| Chemical compound, drug | 2-NBDG | Cayman Chemical | Cat# 11046 | |
| Chemical compound, drug | Cytochalasin B | Wako | Cat# 030–17551 | |
| Chemical compound, drug | Phloretin | TCI chemicals | Cat# P1966 | |
| Chemical compound, drug | 2-morpholinoethanesulfonic acid | Wako | Cat# 341–01622 | |
| Chemical compound, drug | methionine sulfone | Alfa Aesar | Cat# A17027 | |
| Chemical compound, drug | Sodium L-lactate | Sigma Aldrich | Cat# L7022 | |
| Chemical compound, drug | Cholesterol Lipid Concentrate (250 X) | Gibco | Cat# 12531018 | |
| Chemical compound, drug | 100mM-Sodium Pyruvate Solution | Nacalai tesque | Cat# 06977–34 | |
| Chemical compound, drug | Sodium Hydroxide | Wako | Cat# 194–18865 | |
| Chemical compound, drug | 5-fluorouracil | Kyowa Hakko Kirin | N/A | |
| Chemical compound, drug | 2-Deoxy-D-Glucose | Tokyo Chemical Industry Co., Ltd. | Cat# D0051 | |
| Chemical compound, drug | Oligomycin | Cell Signaling Technology | Cat# 9996 L | |
| Chemical compound, drug | FCCP | Sigma Aldrich | Cat# C2920 | |
| Chemical compound, drug | Rotenone | Sigma Aldrich | Cat# R8875 | |
| Chemical compound, drug | Etomoxir (sodium salt) | Cayman chemical | Cat# 11969 | |
| Chemical compound, drug | 6-diazo-5-oxo-L-nor-Leucine | Cayman chemical | Cat# 17580 | |
| Chemical compound, drug | Verapamil | Sigma Aldrich | Cat# V4629 | |
| Chemical compound, drug | N-acetyl-cysteine | Tokyo Chemical Industry Co., Ltd. | Cat# A0905 | |
| Chemical compound, drug | AZ PFKFB3 26 | R&D systems | Cat# 5675 | |
| Chemical compound, drug | Dorsomorphin dihydrochloride | Santa Cruz Biotechnology | Cat# sc-361173 | |
| Chemical compound, drug | LKB1/AAK1 dual inhibitor | Chem Scene | Cat# CS-0342 | |
| Chemical compound, drug | PKM2 inhibitor(compound 3 k) | Selleck | Cat# S8616 | |
| Chemical compound, drug | Recombinant Murine SCF | PeproTech | Cat# 250–03 | |
| Chemical compound, drug | Recombinant Human TPO | PeproTech | Cat# 300–18 | |
| Chemical compound, drug | α-hemolysin | Sigma Aldrich | Cat# H9395 | |
| Chemical compound, drug | Potassium Chloride | Wako | Cat# 7447-40-7 | |
| Chemical compound, drug | Sodium Chloride | Wako | Cat# 7647-14-5 | |
| Chemical compound, drug | Calcium Nitrate Tetrahydrate | Wako | Cat# 13477-34-4 | |
| Chemical compound, drug | Magnesium Sulfate (Anhydrous) | Wako | Cat# 7487-88-9 | |
| Chemical compound, drug | Sodium Hydrogen Carbonate | Wako | Cat# 144-55-8 | |
| Chemical compound, drug | Disodium Hydrogenphosphate 12-Water | Wako | Cat# 10039-32-4 | |
| Chemical compound, drug | Glutathione reduced form | Tokyo Chemical Industry Co., Ltd. | Cat# G0074 | |
| Chemical compound, drug | Ethylene Glycol Bis(β-aminoethylether)-N,N,N',N'-tetraacetic Acid | Nacalai tesque | Cat# 15214–21 | |
| Chemical compound, drug | HEPES | Wako | Cat# 7365-45-9 | |
| Chemical compound, drug | Magnesium Chloride | Wako | Cat# 7786-30-3 | |
| Chemical compound, drug | Adenosine 5’-triphosphate magnesium salt | Sigma Aldrich | Cat# A9187 | |
| Chemical compound, drug | DMSO | Sigma Aldrich | Cat# D8418 | |
| Chemical compound, drug | Ethanol | Nacalai tesque | Cat# 14712–63 | |
| Chemical compound, drug | Methanol | Nacalai tesque | Cat# 21914–03 | |
| Chemical compound, drug | Chloroform | Nacalai tesque | Cat# 08401–65 | |
| Chemical compound, drug | Hoechst 33432 | Thermo Fisher Scientific | Cat# H3570 | (10 µg/mL) |
| Chemical compound, drug | Propidium iodide | Thermo Fisher Scientific | Cat# P3566 | (1:1000) |
| Chemical compound, drug | Flow-Check Fluorspheres | Beckman Coulter | Cat# 7547053 | |
| Chemical compound, drug | TrueCut Cas9 Protein v2 | Thermo Fisher Scientific | Cat# A36498 | |
| Chemical compound, drug | ExTaq | Takara bio | Cat# RR001 | |
| Chemical compound, drug | NotI | Nippon Gene | Cat# 312–01453 | |
| Chemical compound, drug | EcoRI | Nippon Gene | Cat# 314–00112 | |
| Chemical compound, drug | RetroNectin (Recombinant Human Fibronectin Fragment) | Takara | Cat# T100A | |
| Chemical compound, drug | UltraPure DNase_RNase-Free Distilled Water | Invitrogen | Cat# 10977015 | |
| Chemical compound, drug | GSK3368715 | MedChemExpress | Cat# HY-128717A | |
| Commercial assay or kit | RNeasy Mini Kit | QIAGEN | Cat# 74104 | |
| Commercial assay or kit | SuperScript VILO | Thermo Fisher Scientific | Cat# 11754–050 | |
| Commercial assay or kit | 2-mercapto ethanol | Sigma Aldrich | Cat# M6250 | |
| Commercial assay or kit | Flow Cytometry Size Calibration Kit (nonfluorescent microspheres) | Invitrogen | Cat# F13838 | |
| Commercial assay or kit | ‘’Cellno’’ ATP assay reagent Ver.2 | Toyo B-Net Corporation | CA2-50 | |
| Commercial assay or kit | Fixation and Permeabilization Solution | BD Biosciences | Cat# 554722 | |
| Commercial assay or kit | Perm/Wash Buffer | BD Biosciences | Cat# 554723 | |
| Commercial assay or kit | CellROX Deep Red Reagent | Invitrogen | Cat# C10422 | |
| Commercial assay or kit | SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing | Clontech | Cat# Z4888N | |
| Commercial assay or kit | NEBNext Ultra DNA Library Prep Kit for Illumina | New England BioLabs | Cat# E7370S | |
| Commercial assay or kit | CUGA7 gRNA Synthesis Kit | Nippon Gene | Cat# 314–08691 | |
| Commercial assay or kit | Extract-N-Amp Blood PCR Kit | Merck | Cat# XNAB2-1KT | |
| Commercial assay or kit | Wizard SV Gel and PCR Clean-Up System | Promega | Cat# A9281 | |
| Commercial assay or kit | BD Pharmingen FITC BrdU Flow Kit | BD Biosciences | Cat# 559619 | |
| Commercial assay or kit | BD Pharmingen PE Annexin V Apoptosis Detection Kit I | BD Biosciences | Cat# 559763 | |
| Commercial assay or kit | FAOBlue | Funakoshi | Cat# FDV-0033 | |
| Software, algorithm | R v3.5.2 | R Development Core Team, 2018 | http://www.r-project.org | |
| Software, algorithm | TopHat v2.0.13 | 10.1186/gb-2013-14-4-r36; Kim et al., 2013 | https://ccb.jhu.edu/software/tophat/index.shtml | |
| Software, algorithm | Cufflinks v2.2.1 | 10.1038/nbt.1621; Trapnell et al., 2012 | http://cole-trapnell-lab.github.io/cufflinks/ | |
| Software, algorithm | GSEA software v4.3.0 | Broad Institute; Subramanian et al., 2005 | https://www.gsea-msigdb.org/gsea/index.jsp | |
| Software, algorithm | FlowJo version 9 | BD Biosciences | https://www.flowjo.com/ | |
| Software, algorithm | TIDE v3.3.0 | DOI:10.1093/nar/gku93; Brinkman et al., 2014 | https://tide.nki.nl/ | |
| Software, algorithm | OpenMebius | DOI:10.1155/2014/627014; Kajihata et al., 2014 | http://www-shimizu.ist.osaka-u.ac.jp/hp/en/software/OpenMebius.html |
Additional files
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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
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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
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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
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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
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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
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Supplementary file 6
Primer list for genotyping PCR.
- https://cdn.elifesciences.org/articles/87674/elife-87674-supp6-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/87674/elife-87674-mdarchecklist1-v1.docx
