(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) …
Raw data for Figure 1B–H, K and L.
(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 …
Raw data for Figure 1—figure supplement 1D–F, H, K-N.
(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 …
Raw data for Figure 1—figure supplement 2A–H.
(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.
Raw data for Figure 1—figure supplement 3A–D.
(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…
Raw data for Figure 2B–H.
(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 …
Raw data for Figure 3D–L.
(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 …
Raw data for Figure 3—figure supplement 1D–U.
(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), …
Raw data for Figure 3—figure supplement 2B–J.
(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 …
Raw data for Figure 4D–F, I and J.
(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 …
Raw data for Figure 4—figure supplement 1B, C and I.
(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 …
Raw data for Figure 4—figure supplement 2C, D, G-I, K, M, N and P.
(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 …
Raw data for Figure 5D, E, H, I, K, L and M.
(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, …
Raw data for Figure 5—figure supplement 1F,J-L, Q.
(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 …
Raw data for Figure 6A–J.
Data are presented as the mean ± SD. * p≤0.05, ** p≤0.01, *** p≤0.001 as determined using a Student’s t-test.
Raw data for Figure 6—figure supplement 1.
(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 …
Raw data for Figure 7B, C, E, I–K and N.
(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 …
Raw data for Figure 7—figure supplement 1B–D, G, H.
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 |
Custom RPMI medium for culture and ATP analysis.
Composition of custom RPMI medium for culture (upper) and ATP analysis (lower). “-“ means 0 mg/L.
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.
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.
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).
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.
Primer list for genotyping PCR.