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 | 10.1093/nar/gku93; Brinkman et al., 2014 | https://tide.nki.nl/ | |
| Software, algorithm | OpenMebius | 10.1155/2014/627014; Kajihata et al., 2014 | http://www-shimizu.ist.osaka-u.ac.jp/hp/en/software/OpenMebius.html |