MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia
- Department of Medicine, Brigham and Women’s Hospital, United States
- Department of Medicine, Harvard Medical School, United States
- Department of Medicine, Massachusetts General Hospital, United States
- Center for Systems Biology, School of Health Sciences, University of Iceland, Iceland
- Departments of Chemical & Biomolecular Engineering and Molecular Physiology & Biophysics, Vanderbilt University, United States
Figures
Figure 1 with 4 supplements
Effects of 0.5% oxygen on extracellular metabolite fluxes in lung fibroblasts.
(A) Lung fibroblasts (LFs) were cultured in 21% or 0.5% oxygen beginning 24 hr prior to time 0. Samples were collected every 24 hr for 72 hr. (B) Growth curves of LFs in each experimental condition …
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Figure 1—source data 1
Uncropped blot images for Figure 1.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig1-data1-v1.zip
Figure 1—figure supplement 1
Supporting data for extracellular flux calculations.
(A) Cell viability as assessed by live/dead cell staining with acridine orange plus propidium iodide did not differ between 21% and 0.5% oxygen culture conditions (n=3 technical replicates). (B) …
Figure 1—figure supplement 2
Quantifying lactate efflux generated by [U-13C6]-glucose.
Cells were cultured in MCDB131 supplemented with 8 mM [U-13C6]-glucose. Conditioned medium was collected, spiked with [D8]-ᴅʟ-valine internal standard, and analyzed by LC-MS. (A) A standard curve …
Figure 1—figure supplement 3
Effects of 0.2% oxygen on extracellular metabolite fluxes in lung fibroblasts.
(A) Lung fibroblasts (LFs) were cultured in 21% or 0.2% oxygen beginning 24 hr prior to time 0. Samples were collected every 24 hr for 72 hr. (B) Growth curves of LFs in each experimental condition …
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Figure 1—figure supplement 3—source data 1
Uncropped blot images for Figure 1—figure supplement 3.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig1-figsupp3-data1-v1.zip
Figure 1—figure supplement 4
Effects of 0.5% oxygen on extracellular metabolite fluxes in pulmonary artery smooth muscle cells.
(A) Pulmonary artery smooth muscle cells (PASMCs) were cultured in 21% or 0.5% oxygen beginning 24 hr prior to time 0. Samples were collected every 24 hr for 72 hr. (B) Growth curves of LFs in each …
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Figure 1—figure supplement 4—source data 1
Uncropped blot images for Figure 1—figure supplement 4.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig1-figsupp4-data1-v1.zip
Figure 2
Effects of pharmacologic prolyl hydroxylase inhibition on extracellular metabolite fluxes in lung fibroblasts.
(A) Lung fibroblasts (LFs) were treated with the prolyl hydroxlyase inhibitor molidustat (BAY, 10 μM) or DMSO beginning 24 hr prior to time 0. Samples were collected every 24 hr for 72 hr. (B) …
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Figure 2—source data 1
Uncropped blot images for Figure 2.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig2-data1-v1.zip
Figure 3
Cell growth rates following substrate deprivation.
(A, B) Lung fibroblasts (n=8 biological replicates) (A) and pulmonary artery smooth muscle cells (n=4 biological replicates) (B) were cultured in MCDB131 medium lacking either glucose (-GLC) or …
Figure 4 with 2 supplements
Stable isotope labeling of lung fibroblasts following hypoxic and pharmacologic PHD inhibition.
(A) Mass isotopomer distribution (MID) of pyruvate (PYR) following 72 hr labeling with [U-13C6]-glucose (GLC). (B) MID of citrate after 72 hr labeling with [U-13C6]-GLC (C) MID of citrate after 72 …
Figure 4—figure supplement 1
Mass isotopomer distributions after 72 hr of labeling in lung fibroblasts.
Lung fibroblasts (LFs) were labeled with the indicated tracers and intracellular metabolites were analyzed by LC-MS after 72 hr. Mass isotopomer distributions were adjusted for natural abundance. …
Figure 4—figure supplement 2
Mass isotopomer distributions after 36 hr of labeling in pulmonary artery smooth muscle cells.
Pulmonary artery smooth muscle cells (PASMCs) were labeled with the indicated tracers and intracellular metabolites were analyzed by LC-MS after 36 hr. Mass isotopomer distributions were adjusted …
Figure 5 with 5 supplements
Metabolic flux analysis of lung fibroblasts following hypoxic and pharmacologic PHD inhibition.
(A) Ratio of modeled metabolic fluxes in 0.5% oxygen compared to 21% oxygen. Fluxes with non-overlapping confidence intervals are highlighted with arrows colored according to the magnitude of the …
Figure 5—figure supplement 1
Isotope incorporation over the labeling time course.
LFs were cultured in 21% or 0.5% oxygen and labeled with the indicated tracers. Intracellular metabolites were analyzed by LC-MS (FBP, fructose-bisphosphate; PYR, pyruvate; CIT, citrate; MAL, …
Figure 5—figure supplement 2
Isotopically non-stationary metabolic flux analysis of cell metabolism in 21% oxygen.
(A) Metabolic flux model of LF metabolism in 21% oxygen. Arrows are colored by log10(flux). (B) Metabolic flux model of PASMC metabolism in 21% oxygen as in (A).
Figure 5—figure supplement 3
Comparison of lung fibroblast and pulmonary artery smooth muscle cell metabolic fluxes.
Ratio of metabolic fluxes in PASMCs compared to LFs. Fluxes with non-overlapping confidence intervals are highlighted with arrows colored according to the magnitude of the change. Arrow thickness …
Figure 5—figure supplement 4
Comparison of pulmonary artery smooth muscle cell metabolic fluxes in 21% and 0.5% oxygen.
Ratio of modeled metabolic fluxes in 0.5% oxygen compared to 21% oxygen. Fluxes with non-overlapping confidence intervals are highlighted with arrows colored according to the magnitude of the fold …
Figure 5—figure supplement 5
Growth rate adjusted changes in hypoxic lung fibroblast metabolism.
LF fluxes were normalized to cell growth rate. Graph depicts the ratio of normalized metabolic fluxes in LFs cultured in 0.5% oxygen compared to 21% oxygen control. Fluxes with non-overlapping …
Figure 6
Hypoxia increases reductive carboxylation in pulmonary artery smooth muscle cells.
(A) Reductive carboxylation describes the converstion of α-ketoglutarate (AKG) to isocitrate by reverse flux through isocitrate dehydrogenase (IDH) (blue arrow). This yields M+5 citrate. (B) …
Figure 7
PHD inhibition increases lactate uptake and oxidation.
(A) Exchange flux estimates for lactate import presented as in (A). (C) Mass isotopomer distributions of lactate (LAC), fructose-bisphosphate (FBP), pyruvate (PYR), citrate (CIT), α-ketoglutarate …
Figure 8 with 1 supplement
Hypoxia prevents BAY-stimulated increases in glycolysis.
(A) LFs were cultured in standard growth medium and treated with molidustat (BAY, 10 μM) or vehicle (DMSO, 0.1%) in 21% or 0.5% oxygen conditions for 72 hr. Growth rates were determined by linear …
Figure 8—figure supplement 1
Hypoxia attenuates siPHD2-stimulated increases in glycolysis.
LFs were treated with siRNA to knock down PHD2 expression. (A) Cell growth was not impacted by siPHD2 treatment. (B) Representative immunoblot of LF cell lysates at 72 hr demonstrating substantial …
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Figure 8—figure supplement 1—source data 1
Uncropped blot images for Figure 8—figure supplement 1.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig8-figsupp1-data1-v1.zip
Figure 9 with 2 supplements
Metabolomic analysis of molidustat treatment in normoxia and hypoxia.
(A) Principal component analysis of intracellular metabolites following 72 hr of treatment suggests a dominant effect of hypoxia over pharmacologic PHD inhibition on the metabolome (n=7). (B) …
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Figure 9—source data 1
Source data for Figure 9C.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig9-data1-v1.csv
Figure 9—figure supplement 1
Metabolomic profiling of hypoxia and BAY treated lung fibroblasts.
(A) Volcano plot of differentially regulated metabolites following 0.5% oxygen culture. (B) Volcano plot of differentially regulated metabolites by BAY treatment in 21% oxygen. (C) Venn diagram …
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Figure 9—figure supplement 1—source data 1
Source data for Figure 9—figure supplement 1D.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig9-figsupp1-data1-v1.csv
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Figure 9—figure supplement 1—source data 2
Source data for Figure 9—figure supplement 1E.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig9-figsupp1-data2-v1.csv
Figure 9—figure supplement 2
Changes in NAD(H) and NADP(H) following hypoxia and BAY treamtent.
(A) NAD+, (B) NADH, and (C) NADH/NAD+ from LFs cultured for 72 hr in 0.5% oxygen ±BAY (n=5).(D) NADP+, (E) NADPH, and (F) NADPH/NADP+ from LFs cultured for 72 hr in 0.5% oxygen ±BAY (n=4). Data are …
Figure 10 with 2 supplements
Transcriptomic analysis of molidustat treatment in normoxia and hypoxia.
(A) Principal component analysis of transcriptional changes in lung fibroblasts following 72 hr of treatment with 0.5% oxygen or molidustat (BAY), separately or together (n=4). (B) Volcano plot …
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Figure 10—source data 1
Source data for Figure 10C.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig10-data1-v1.csv
Figure 10—figure supplement 1
Transcriptomic profiling of hypoxia and BAY-treated lung fibroblasts.
(A) Volcano plot of differentially expressed genes following 0.5% oxygen culture. (B) Volcano plot of differentially expressed genes following BAY treatment. (C) Venn diagram illustrating the number …
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Figure 10—figure supplement 1—source data 1
Source data for Figure 10—figure supplement 1E.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig10-figsupp1-data1-v1.csv
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Figure 10—figure supplement 1—source data 2
Source data for Figure 10—figure supplement 1F.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig10-figsupp1-data2-v1.csv
Figure 10—figure supplement 2
CDKN1A expression in BAY- and siPHD2-treated cells.
(A) Normalized mRNA counts from RNA-seq analysis of LFs treated with hypoxia ±BAY (n=4). (B) Relative CDKN1A expression by RT-qPCR in siPHD2-treated in normoxia and hypoxia (n=4). Data are mean ± …
Figure 11 with 2 supplements
MYC regulates HIF-dependent glycolytic flux.
(A) Representative immunoblot of MYC protein expression in lung fibroblasts (LFs) following 72 hr of treatment with 0.5% oxygen or molidustat BAY, (B). (B) Quantification of band densities from (A). …
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Figure 11—source data 1
Uncropped blot images for Figure 11A.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig11-data1-v1.zip
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Figure 11—source data 2
Uncropped blot images for Figure 11D and G.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig11-data2-v1.zip
Figure 11—figure supplement 1
Effects of hypoxia on MYC protein in siPHD2-treated cells.
(A) Representative immunoblot of MYC protein levels in LFs treated with siPHD2 (P) or siCTL (C) and exposed to 0.5% oxygen for 72 hr. (B) Quantification of MYC band densities as in (A). Data are …
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Figure 11—figure supplement 1—source data 1
Uncropped blot images for Figure 11—figure supplement 1.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig11-figsupp1-data1-v1.zip
Figure 11—figure supplement 2
Effects of MYC on HIF stabilization and target gene exprssion.
(A) Representative immunoblot of HIF-1α and LDHA protein levels in LFs treated with siMYC (M) or siCTL (C) and exposed to 0.5% oxygen for 72 hr. (B) Quantification of HIF-1α band densities as in (A).…
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Figure 11—figure supplement 2—source data 1
Uncropped blot images for Figure 11—figure supplement 2.
- https://cdn.elifesciences.org/articles/82597/elife-82597-fig11-figsupp2-data1-v1.zip
Figure 12
Total DNA as a surrogate for cell count.
(A) Standard curve of lung fibroblast (LF) cell count v. total DNA by PicoGreen measurement used to interpolate cell numbers from DNA measurements. Data are mean ± SEM of three biological …
Author response image 1
Tables
Table 1
LF fluxes in 21% and 0.5% oxygen.
| Type | Pathway | ID | Reaction | 21% * | 0.5%† | Ratio | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| Flux | LB | UB | Flux | LB | UB | |||||
| NET | Transport | GLUT | GLC.x → GLC | 5.14E+02 | 5.11E+02 | 5.21E+02 | 4.41E+02 | 4.26E+02 | 4.58E+02 | 0.86 |
| PYRR | PYR.x → PYR.c | 7.56E+01 | 7.31E+01 | 7.96E+01 | 6.21E+01 | 5.83E+01 | 6.60E+01 | 0.82 | ||
| MCT | LAC ↔ LAC.x | 9.99E+02 | 9.98E+02 | 1.02E+03 | 8.91E+02 | 8.62E+02 | 9.25E+02 | 0.89 | ||
| ALAR | ALA → ALA.x | 2.25E+00 | 1.95E+00 | 2.49E+00 | 5.84E-01 | 1.10E-03 | 1.16E+00 | 0.26 | ||
| GLNR | GLN.x → GLN | 4.15E+01 | 4.06E+01 | 4.16E+01 | 1.43E+01 | 1.26E+01 | 1.94E+01 | 0.34 | ||
| GLUR | GLU ↔ GLU.x | 1.62E+01 | 1.58E+01 | 1.68E+01 | 7.55E+00 | 6.88E+00 | 8.15E+00 | 0.47 | ||
| ASPR | ASP → ASP.x | 2.57E+00 | 2.53E+00 | 2.68E+00 | 1.08E+00 | 4.17E-01 | 1.69E+00 | 0.42 | ||
| SERR | SER.x → SER | 1.42E+01 | 1.35E+01 | 1.49E+01 | 5.49E+00 | 4.99E+00 | 6.06E+00 | 0.39 | ||
| CYSR | CYX.x → CYS +CYS | 4.41E+00 | 4.23E+00 | 4.58E+00 | 1.65E+00 | 1.32E+00 | 2.08E+00 | 0.37 | ||
| GLYR | GLY → GLY.x | 2.05E+00 | 1.90E+00 | 2.15E+00 | 2.60E-01 | 2.00E-02 | 4.92E-01 | 0.13 | ||
| Glycolysis | HK | GLC → G6P | 5.14E+02 | 5.11E+02 | 5.21E+02 | 4.41E+02 | 4.26E+02 | 4.58E+02 | 0.86 | |
| PGI | G6P ↔ F6P | 5.11E+02 | 4.99E+02 | 5.24E+02 | 4.23E+02 | 4.04E+02 | 4.40E+02 | 0.83 | ||
| PFK | F6P → FBP | 5.09E+02 | 5.00E+02 | 5.12E+02 | 4.32E+02 | 4.17E+02 | 4.49E+02 | 0.85 | ||
| ALDO | FBP ↔ DHAP +GAP | 5.09E+02 | 5.00E+02 | 5.12E+02 | 4.32E+02 | 4.17E+02 | 4.49E+02 | 0.85 | ||
| TPI | DHAP ↔ GAP | 5.08E+02 | 5.06E+02 | 5.08E+02 | 4.31E+02 | 4.15E+02 | 4.48E+02 | 0.85 | ||
| GAPDH | GAP ↔ 3 PG | 1.02E+03 | 9.96E+02 | 1.04E+03 | 8.69E+02 | 8.35E+02 | 9.03E+02 | 0.85 | ||
| ENO | 3 PG → PEP | 1.01E+03 | 9.99E+02 | 1.03E+03 | 8.68E+02 | 8.36E+02 | 9.00E+02 | 0.86 | ||
| PK | PEP → PYR.c | 1.04E+03 | 9.95E+02 | 1.04E+03 | 8.78E+02 | 8.36E+02 | 9.21E+02 | 0.84 | ||
| LDH | PYR.c ↔ LAC | 9.99E+02 | 9.98E+02 | 1.02E+03 | 8.91E+02 | 8.62E+02 | 9.25E+02 | 0.89 | ||
| GPT1 | PYR.c ↔ ALA | 1.19E+01 | 9.12E+00 | 1.19E+01 | 5.55E+00 | –9.08E+02 | 6.13E+00 | 0.47 | ||
| GPT2 | PYR.m ↔ ALA | –2.58E+00 | –4.56E+00 | 2.87E+00 | –2.40E-03 | –3.22E+01 | 9.11E+02 | |||
| Pentose phosphate pathway | G6PD | G6P → P5P+CO2 | 1.26E-07 | 0.00E+00 | 3.91E-01 | 1.62E+01 | 4.41E+00 | 2.89E+01 | 128571428.57 | |
| TK1 | P5P+P5 P ↔ S7P+GAP | –9.11E-01 | –9.29E-01 | –8.30E-01 | 4.76E+00 | –1.22E-01 | 9.62E+00 | –5.23 | ||
| TA | S7P+GAP ↔ F6P+E4 P | –9.11E-01 | –9.29E-01 | –8.30E-01 | 4.76E+00 | –1.22E-01 | 9.62E+00 | –5.23 | ||
| TK2 | P5P+E4 P ↔ F6P+GAP | –9.11E-01 | –9.29E-01 | –8.30E-01 | 4.76E+00 | –1.22E-01 | 9.62E+00 | –5.23 | ||
| Anaplerosis | PYRT | PYR.c → PYR.m | 1.16E+02 | 1.16E+02 | 1.19E+02 | 4.42E+01 | 3.82E+01 | 9.58E+02 | ||
| PC | PYR.m+CO2 → OAC | 1.88E+01 | 1.74E+01 | 1.91E+01 | 1.37E+01 | 9.82E+00 | 2.69E+01 | |||
| PEPCK | OAC → PEP +CO2 | 2.56E+01 | 1.58E+01 | 2.57E+01 | 9.66E+00 | 0.00E+00 | 2.60E+01 | |||
| ME2 | MAL → PYR.m +CO2 | 2.05E+00 | 9.51E-02 | 2.68E+00 | 1.00E-07 | 0.00E+00 | 2.25E+01 | |||
| ME1 | MAL → PYR.c +CO2 | 2.78E-02 | 0.00E+00 | 2.63E+01 | 8.71E-05 | 0.00E+00 | 2.52E+01 | |||
| FAO | FAO → AcCoA.m | 1.00E-07 | 0.00E+00 | 2.13E+00 | 6.58E-06 | 0.00E+00 | 7.73E-01 | |||
| GLDH | GLU ↔ AKG | 1.71E+01 | 1.56E+01 | 1.84E+01 | 9.11E-01 | –6.16E-01 | 7.27E+00 | 0.05 | ||
| GLS | GLN ↔ GLU | 3.78E+01 | 3.60E+01 | 3.86E+01 | 1.17E+01 | 1.01E+01 | 1.70E+01 | 0.31 | ||
| Tricarboxylic acid cycle | PDH | PYR.m → AcCoA.m +CO2 | 1.02E+02 | 8.76E+01 | 1.15E+02 | 3.05E+01 | 2.86E+01 | 5.24E+01 | 0.3 | |
| CS | AcCoA.m+OAC → CIT | 1.02E+02 | 8.30E+01 | 1.11E+02 | 3.05E+01 | 2.88E+01 | 5.09E+01 | 0.3 | ||
| IDH | CIT ↔ AKG +CO2 | 2.49E+01 | 2.42E+01 | 2.53E+01 | 1.01E+01 | 8.75E+00 | 1.41E+01 | 0.41 | ||
| OGDH | AKG → SUC +CO2 | 4.19E+01 | 4.01E+01 | 4.25E+01 | 1.10E+01 | 7.87E+00 | 2.02E+01 | 0.26 | ||
| SDH | SUC ↔ FUM | 4.19E+01 | 4.01E+01 | 4.25E+01 | 1.10E+01 | 7.87E+00 | 2.02E+01 | 0.26 | ||
| FH | FUM ↔ MAL | 4.19E+01 | 4.01E+01 | 4.25E+01 | 1.10E+01 | 7.87E+00 | 2.02E+01 | 0.26 | ||
| MDH | MAL ↔ OAC | 1.17E+02 | 1.08E+02 | 1.24E+02 | 3.14E+01 | 2.62E+01 | 5.70E+01 | 0.27 | ||
| GOT | OAC ↔ ASP | 8.11E+00 | 8.06E+00 | 8.23E+00 | 4.98E+00 | 4.32E+00 | 5.64E+00 | 0.61 | ||
| Amino acid metabolism | PST | 3 PG → SER | 1.95E+00 | 1.63E+00 | 2.00E+00 | 2.42E-01 | 1.34E-01 | 3.57E+01 | ||
| SHT | SER ↔ GLY +MEETHF | 6.38E+00 | 6.22E+00 | 6.43E+00 | 3.91E+00 | 3.71E+00 | 4.10E+00 | 0.61 | ||
| CYST | SER ↔ CYS | –7.12E+00 | –7.19E+00 | –6.81E+00 | –2.10E+00 | –2.97E+00 | –1.44E+00 | 0.3 | ||
| SD | SER → PYR.c | 1.17E+01 | 1.04E+01 | 1.20E+01 | 2.82E-01 | 0.00E+00 | 1.47E+00 | 0.02 | ||
| GLYS | CO2 +MEETHF → GLY | 3.39E+00 | 3.35E+00 | 3.49E+00 | 1.80E+00 | 1.66E+00 | 1.93E+00 | 0.53 | ||
| Biomass | BIOMASS | 1216*AcCoA.c+295.6 *ALA +232.4*ASP +114.7*CO2 +71.43*CYS +57.14*DHAP +142.4*G6P+158.6 *GLN +190.1*GLU +324.2*GLY +125.6*MEETHF +114.7*P5P+217.2*SER → biomass | 2.38E-02 | 2.34E-02 | 2.39E-02 | 1.68E-02 | 1.61E-02 | 1.75E-02 | 0.71 | |
| ACL | CIT → AcCoA.c +MAL | 7.74E+01 | 6.29E+01 | 1.04E+02 | 2.04E+01 | 1.95E+01 | 3.71E+01 | 0.26 | ||
| LIPS | AcCoA.c → lipid | 4.84E+01 | 4.55E+01 | 4.84E+01 | 1.00E-07 | 0.00E+00 | 1.68E+01 | 0 | ||
| Mixing | cPYR | 0*PYR.c → PYR.ms | 1.00E+00 | 8.47E-01 | 1.00E+00 | 1.42E-01 | 0.00E+00 | 1.00E+00 | ||
| mPYR | 0*PYR.m → PYR.ms | 1.00E-07 | 0.00E+00 | 1.53E-01 | 8.58E-01 | 0.00E+00 | 1.00E+00 | |||
| sPYR | PYR.ms → PYR.fix | 1.00E+00 | 1.00E+00 | 1.00E+00 | 1.00E+00 | 1.00E+00 | 1.00E+00 | |||
| EXCH | Transport | MCT | LAC ↔ LAC.x | 1.00E-07 | 0.00E+00 | 1.05E-01 | 1.52E+03 | 1.35E+03 | 2.41E+03 | 15200000000 |
| GLUR | GLU ↔ GLU.x | 5.10E+00 | 4.77E+00 | 5.23E+00 | 1.54E+00 | 1.11E+00 | 2.54E+00 | 0.3 | ||
| Glycolysis | PGI | G6P ↔ F6P | 2.78E+05 | 1.77E+05 | Inf | 2.46E+05 | 0.00E+00 | Inf | ||
| ALDO | FBP ↔ DHAP +GAP | 1.43E+02 | 1.43E+02 | 1.43E+02 | 3.20E+02 | 2.79E+02 | 3.60E+02 | 2.24 | ||
| TPI | DHAP ↔ GAP | 4.33E+03 | 4.33E+03 | 1.09E+04 | 1.70E+03 | 1.06E+03 | 3.06E+03 | 0.39 | ||
| GAPDH | GAP ↔ 3 PG | 4.42E+02 | 4.72E+00 | 4.50E+02 | 1.00E-07 | 0.00E+00 | 2.39E+02 | |||
| LDH | PYR.c ↔ LAC | 1.63E+03 | 1.62E+03 | 1.80E+03 | 4.80E+00 | 0.00E+00 | 3.51E+02 | 0 | ||
| GPT1 | PYR.c ↔ ALA | 1.00E-07 | 0.00E+00 | 2.61E-01 | 8.32E+02 | 0.00E+00 | 9.06E+02 | |||
| GPT2 | PYR.m ↔ ALA | 4.21E-04 | 0.00E+00 | 2.92E+00 | 1.28E-04 | 0.00E+00 | ||||
| Pentose phosphate pathway | TK1 | P5P+P5 P ↔ S7P+GAP | 9.97E+04 | 6.27E+03 | Inf | 1.47E+02 | 6.67E+01 | 2.60E+02 | 0 | |
| TA | S7P+GAP ↔ F6P+E4 P | 5.93E+00 | 5.79E+00 | 6.97E+00 | 2.35E-04 | 0.00E+00 | 7.54E+00 | |||
| TK2 | P5P+E4 P ↔ F6P+GAP | 1.00E+07 | -Inf | Inf | 9.05E+00 | 4.10E+00 | 1.43E+01 | |||
| Anaplerosis | GLDH | GLU ↔ AKG | 1.52E+03 | 1.52E+03 | 7.13E+03 | 3.78E+02 | 1.93E+02 | 1.94E+03 | ||
| GLS | GLN ↔ GLU | 3.99E-01 | 0.00E+00 | 8.04E-01 | 1.00E-07 | 0.00E+00 | 3.84E-01 | |||
| Tricarboxylic acid cycle | IDH | CIT ↔ AKG +CO2 | 4.55E+00 | 4.03E+00 | 5.19E+00 | 2.52E+00 | 1.80E+00 | 4.50E+00 | ||
| SDH | SUC ↔ FUM | 1.22E+03 | Inf | 7.60E+01 | 2.57E+01 | Inf | ||||
| FH | FUM ↔ MAL | 3.66E+05 | 1.95E+05 | Inf | 5.05E+05 | 3.06E+02 | Inf | |||
| MDH | MAL ↔ OAC | 1.11E+03 | 7.88E+02 | 2.38E+03 | 1.33E+02 | 7.22E+01 | 3.25E+02 | 0.12 | ||
| GOT | OAC ↔ ASP | 1.00E+07 | -Inf | Inf | 4.42E+01 | 0.00E+00 | Inf | |||
| Amino acid metabolism | SHT | SER ↔ GLY +MEETHF | 5.10E+00 | 8.92E-01 | 5.25E+00 | 6.07E-07 | 0.00E+00 | 3.32E+02 | ||
| CYST | SER ↔ CYS | 1.52E-05 | 0.00E+00 | 2.55E-04 | 1.46E-02 | 0.00E+00 | Inf | |||
-
*
SSR 391.7 [311.2‐416.6] (95% CI, 362 DOF).
-
†
SSR 334.3 [311.2‐416.6] (95% CI, 362 DOF).
Table 2
LF fluxes following DMSO and BAY treatment.
| Type | Pathway | ID | Reaction | DMSO* | BAY† | Ratio | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| Flux | LB | UB | Flux | LB | UB | |||||
| NET | Transport | GLUT | GLC.x → GLC | 6.12E+02 | 6.12E+02 | 6.12E+02 | 8.80E+02 | 8.80E+02 | 8.80E+02 | 1.44 |
| PYRR | PYR.x → PYR.c | 9.98E+01 | 9.95E+01 | 1.01E+02 | 6.06E+01 | 6.06E+01 | 6.06E+01 | 0.61 | ||
| MCT | LAC ↔ LAC.x | 8.19E+02 | 8.17E+02 | 8.20E+02 | 1.33E+03 | 1.33E+03 | 1.33E+03 | 1.62 | ||
| ALAR | ALA → ALA.x | 2.67E+00 | 2.36E+00 | 3.29E+00 | 5.98E+00 | 5.88E+00 | 6.24E+00 | 2.24 | ||
| GLNR | GLN.x → GLN | 3.78E+01 | 3.77E+01 | 3.79E+01 | 2.06E+01 | 2.06E+01 | 2.06E+01 | 0.54 | ||
| GLUR | GLU ↔ GLU.x | 1.61E+01 | 1.56E+01 | 1.62E+01 | 1.68E+01 | 1.68E+01 | 1.68E+01 | 1.05 | ||
| ASPR | ASP → ASP.x | 2.36E+00 | 2.32E+00 | 2.49E+00 | 1.80E+00 | 1.80E+00 | 1.81E+00 | 0.76 | ||
| SERR | SER.x → SER | 1.03E+01 | 1.03E+01 | 1.06E+01 | 2.50E+00 | 2.50E+00 | 2.50E+00 | 0.24 | ||
| CYSR | CYX.x → CYS+CYS | 2.79E+00 | 2.79E+00 | 2.95E+00 | 3.07E-01 | 3.06E-01 | 3.07E-01 | 0.11 | ||
| GLYR | GLY → GLY.x | 2.52E+00 | 2.30E+00 | 2.73E+00 | 5.52E-01 | 4.30E-01 | 7.45E-01 | 0.22 | ||
| Glycolysis | HK | GLC → G6P | 6.12E+02 | 6.12E+02 | 6.12E+02 | 8.80E+02 | 8.80E+02 | 8.80E+02 | 1.44 | |
| PGI | G6P ↔ F6P | 6.09E+02 | 6.08E+02 | 6.09E+02 | 8.42E+02 | 8.42E+02 | 8.42E+02 | 1.38 | ||
| PFK | F6P → FBP | 6.07E+02 | 6.07E+02 | 6.07E+02 | 8.65E+02 | 8.65E+02 | 8.65E+02 | 1.43 | ||
| ALDO | FBP ↔ DHAP+GAP | 6.07E+02 | 6.07E+02 | 6.07E+02 | 8.65E+02 | 8.65E+02 | 8.65E+02 | 1.43 | ||
| TPI | DHAP ↔ GAP | 6.06E+02 | 6.06E+02 | 6.06E+02 | 8.65E+02 | 8.65E+02 | 8.65E+02 | 1.43 | ||
| GAPDH | GAP ↔ 3PG | 1.21E+03 | 1.21E+03 | 1.21E+03 | 1.74E+03 | 1.74E+03 | 1.74E+03 | 1.44 | ||
| ENO | 3PG → PEP | 1.21E+03 | 1.21E+03 | 1.21E+03 | 1.57E+03 | 1.57E+03 | 1.57E+03 | 1.3 | ||
| PK | PEP → PYR.c | 1.23E+03 | 1.19E+03 | 1.23E+03 | 1.65E+03 | 1.65E+03 | 1.65E+03 | 1.34 | ||
| LDH | PYR.c ↔ LAC | 8.19E+02 | 8.17E+02 | 8.20E+02 | 1.33E+03 | 1.33E+03 | 1.33E+03 | 1.62 | ||
| GPT1 | PYR.c ↔ ALA | 9.62E+00 | 9.44E+00 | 9.62E+00 | 9.36E+00 | 9.32E+00 | 9.42E+00 | 0.97 | ||
| GPT2 | PYR.m ↔ ALA | 1.14E-01 | 2.28E-07 | –1.22E-05 | 6.41E-04 | |||||
| Pentose phosphate pathway | G6PD | G6P → P5P+CO2 | 2.02E-02 | 0.00E+00 | 1.08E+00 | 3.64E+01 | 3.64E+01 | 3.64E+01 | 1801.98 | |
| TK1 | P5P+P5P ↔ S7P+GAP | –9.06E-01 | –9.28E-01 | –9.06E-01 | 1.17E+01 | 1.17E+01 | 1.17E+01 | –12.89 | ||
| TA | S7P+GAP ↔ F6P+E4P | –9.06E-01 | –9.28E-01 | –9.06E-01 | 1.17E+01 | 1.17E+01 | 1.17E+01 | –12.89 | ||
| TK2 | P5P+E4P ↔ F6P+GAP | –9.06E-01 | –9.28E-01 | –9.06E-01 | 1.17E+01 | 1.17E+01 | 1.17E+01 | –12.89 | ||
| Anaplerosis | PYRT | PYR.c → PYR.m | 4.99E+02 | 4.97E+02 | 4.99E+02 | 5.50E+02 | 5.50E+02 | 5.50E+02 | 1.1 | |
| PC | PYR.m+CO2 → OAC | 2.11E+01 | 2.07E+01 | 2.17E+01 | 9.05E+01 | 9.05E+01 | 9.05E+01 | 4.28 | ||
| PEPCK | OAC → PEP+CO2 | 1.36E+01 | 1.36E+01 | 1.37E+01 | 8.58E+01 | 8.58E+01 | 8.58E+01 | 6.31 | ||
| ME2 | MAL → PYR.m+CO2 | 1.30E+01 | 1.28E+01 | 1.37E+01 | 1.00E-07 | 0.00E+00 | 9.49E-06 | 0 | ||
| ME1 | MAL → PYR.c+CO2 | 3.20E-03 | 0.00E+00 | 1.73E+00 | 1.00E-07 | 0.00E+00 | 2.15E-05 | |||
| FAO | FAO → AcCoA.m | 1.00E-07 | 0.00E+00 | 3.48E+00 | 1.09E-04 | 8.34E-06 | 4.14E-02 | |||
| GLDH | GLU ↔ AKG | 1.33E+01 | 1.31E+01 | 1.35E+01 | –2.46E-01 | –2.47E-01 | –2.46E-01 | –0.02 | ||
| GLS | GLN ↔ GLU | 3.40E+01 | 3.35E+01 | 3.42E+01 | 1.88E+01 | 1.88E+01 | 1.88E+01 | 0.55 | ||
| Tricarboxylic acid cycle | PDH | PYR.m → AcCoA.m+CO2 | 4.90E+02 | 4.90E+02 | 4.92E+02 | 4.60E+02 | 4.60E+02 | 4.60E+02 | 0.94 | |
| CS | AcCoA.m+OAC → CIT | 4.90E+02 | 4.84E+02 | 4.91E+02 | 4.60E+02 | 4.60E+02 | 4.60E+02 | 0.94 | ||
| IDH | CIT ↔ AKG+CO2 | 2.70E+01 | 2.70E+01 | 2.76E+01 | 1.45E+01 | 1.45E+01 | 1.45E+01 | 0.54 | ||
| OGDH | AKG → SUC+CO2 | 4.03E+01 | 3.99E+01 | 4.04E+01 | 1.43E+01 | 1.43E+01 | 1.43E+01 | 0.35 | ||
| SDH | SUC ↔ FUM | 4.03E+01 | 3.99E+01 | 4.04E+01 | 1.43E+01 | 1.43E+01 | 1.43E+01 | 0.35 | ||
| FH | FUM ↔ MAL | 4.03E+01 | 3.99E+01 | 4.04E+01 | 1.43E+01 | 1.43E+01 | 1.43E+01 | 0.35 | ||
| MDH | MAL ↔ OAC | 4.91E+02 | 4.91E+02 | 4.92E+02 | 4.60E+02 | 4.60E+02 | 4.60E+02 | 0.94 | ||
| GOT | OAC ↔ ASP | 7.91E+00 | 7.76E+00 | 7.98E+00 | 4.46E+00 | 4.46E+00 | 4.46E+00 | 0.56 | ||
| Amino acid metabolism | PST | 3PG → SER | 4.03E-01 | 3.74E-01 | 5.04E-01 | 1.73E+02 | 1.73E+02 | 1.73E+02 | 429.83 | |
| SHT | SER ↔ GLY+MEETHF | 6.63E+00 | 6.59E+00 | 6.65E+00 | 2.85E+00 | 2.79E+00 | 2.93E+00 | 0.43 | ||
| CYST | SER ↔ CYS | –3.88E+00 | –3.91E+00 | –3.87E+00 | 2.03E-01 | 2.02E-01 | 2.03E-01 | –0.05 | ||
| SD | SER → PYR.c | 2.80E+00 | 2.80E+00 | 2.80E+00 | 1.70E+02 | 1.70E+02 | 1.70E+02 | 60.81 | ||
| GLYS | CO2+MEETHF → GLY | 3.63E+00 | 3.50E+00 | 3.65E+00 | 1.41E+00 | 1.30E+00 | 1.46E+00 | 0.39 | ||
| Biomass | BIOMASS | 1216*AcCoA.c+295.6 *ALA +232.4*ASP+114.7*CO2+71.43*CYS+57.14*DHAP+142.4*G6P+ 158.6*GLN+190.1*GLU +324.2*GLY+125.6*MEETHF+114.7*P5P+217.2 *SER → biomass | 2.39E-02 | 2.39E-02 | 2.50E-02 | 1.14E-02 | 1.14E-02 | 1.14E-02 | 0.48 | |
| ACL | CIT → AcCoA.c+MAL | 4.63E+02 | 4.63E+02 | 4.66E+02 | 4.45E+02 | 4.45E+02 | 4.45E+02 | 0.96 | ||
| LIPS | AcCoA.c → lipid | 4.34E+02 | 4.29E+02 | 4.34E+02 | 4.32E+02 | 4.32E+02 | 4.32E+02 | |||
| Mixing | cPYR | 0*PYR.c → PYR.ms | 1.00E+00 | 9.99E-01 | 1.00E+00 | 1.00E-07 | 0.00E+00 | 1.00E+00 | ||
| mPYR | 0*PYR.m → PYR.ms | 1.00E-07 | 0.00E+00 | 9.83E-04 | 1.00E+00 | 0.00E+00 | 1.00E+00 | |||
| sPYR | PYR.ms → PYR.fix | 1.00E+00 | 1.00E+00 | 1.00E+00 | 1.00E+00 | 1.00E+00 | 1.00E+00 | |||
| EXCH | Transport | MCT | LAC ↔ LAC.x | 6.24E-04 | 0.00E+00 | 3.56E+00 | 7.11E+02 | 7.11E+02 | 7.11E+02 | 1139423.08 |
| GLUR | GLU ↔ GLU.x | 5.06E+00 | 4.82E+00 | 5.75E+00 | 3.48E+00 | 3.48E+00 | 3.48E+00 | 0.69 | ||
| Glycolysis | PGI | G6P ↔ F6P | 1.40E+06 | 1.39E+06 | Inf | 4.31E+06 | 4.31E+06 | 4.31E+06 | ||
| ALDO | FBP ↔ DHAP+GAP | 2.38E+02 | 2.38E+02 | 2.38E+02 | 1.02E+03 | 1.02E+03 | 1.02E+03 | 4.28 | ||
| TPI | DHAP ↔ GAP | 9.99E+06 | Inf | 7.57E+03 | 7.57E+03 | 7.57E+03 | ||||
| GAPDH | GAP ↔ 3PG | 5.81E+02 | 5.81E+02 | 7.25E+02 | 1.09E+02 | 1.07E+02 | 1.09E+02 | 0.19 | ||
| LDH | PYR.c ↔ LAC | 2.65E+03 | 2.58E+03 | 2.65E+03 | 4.92E+01 | 4.91E+01 | 4.94E+01 | 0.02 | ||
| GPT1 | PYR.c ↔ ALA | 1.00E-07 | 0.00E+00 | 5.60E-02 | 2.45E+03 | 2.45E+03 | 2.45E+03 | 24500000000 | ||
| GPT2 | PYR.m ↔ ALA | 1.00E-07 | 0.00E+00 | 5.65E-02 | 1.00E-07 | 0.00E+00 | 1.20E-05 | |||
| Pentose phosphate pathway | TK1 | P5P+P5P ↔ S7P+GAP | 1.28E+06 | 9.01E+03 | Inf | 1.00E+07 | -Inf | Inf | ||
| TA | S7P+GAP ↔ F6P+E4P | 8.89E+00 | 8.88E+00 | 9.53E+00 | 5.10E+01 | 5.10E+01 | 5.10E+01 | 5.74 | ||
| TK2 | P5P+E4P ↔ F6P+GAP | 6.93E+00 | 5.12E+00 | 6.98E+00 | 1.00E-07 | 0.00E+00 | 1.56E-04 | 0 | ||
| Anaplerosis | GLDH | GLU ↔ AKG | 5.63E+03 | 4.43E+03 | 5.66E+03 | 1.42E+03 | 1.42E+03 | 1.42E+03 | 0.25 | |
| GLS | GLN ↔ GLU | 1.27E+00 | 1.20E+00 | 1.50E+00 | 5.52E-01 | 5.51E-01 | 5.55E-01 | 0.43 | ||
| Tricarboxylic acid cycle | IDH | CIT ↔ AKG+CO2 | 3.36E+00 | 3.24E+00 | 3.92E+00 | 4.66E+00 | 4.66E+00 | 4.66E+00 | 1.39 | |
| SDH | SUC ↔ FUM | 4.30E+02 | 4.30E+02 | 1.46E+06 | 1.04E+04 | 1.04E+04 | 1.04E+04 | |||
| FH | FUM ↔ MAL | 7.29E+06 | -Inf | Inf | 4.56E+06 | 4.56E+06 | 4.56E+06 | |||
| MDH | MAL ↔ OAC | 5.49E+02 | 5.47E+02 | 5.49E+02 | 1.00E-07 | 0.00E+00 | 6.30E-03 | 0 | ||
| GOT | OAC ↔ ASP | 1.04E+02 | 1.04E+02 | 1.04E+02 | 4.76E+05 | 4.76E+05 | 4.76E+05 | 4576.92 | ||
| Amino acid metabolism | SHT | SER ↔ GLY+MEETHF | 1.39E+00 | 1.37E+00 | 1.41E+00 | 1.86E+03 | 1.86E+03 | 1.86E+03 | 1338.13 | |
| CYST | SER ↔ CYS | 1.25E-07 | 0.00E+00 | 4.22E-02 | 1.33E-01 | 1.33E-01 | 1.33E-01 | 1064000 | ||
-
*
SSR 393.5 [311.2–416.6] (95% CI, 362 DOF).
-
†
SSR 392.4 [308.4–413.4] (95% CI, 359 DOF).
Table 3
PASMC fluxes in 21% and 0.5% oxygen.
| Type | Pathway | ID | Reaction | 21%* | 0.5%† | Ratio | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| Flux | LB | UB | Flux | LB | UB | |||||
| NET | Transport | GLUT | GLC.x → GLC | 4.28E+02 | 4.28E+02 | 4.28E+02 | 3.65E+02 | 3.65E+02 | 3.65E+02 | 0.85 |
| PYRR | PYR.x → PYR.c | 1.04E+02 | 1.02E+02 | 1.09E+02 | 4.53E+01 | 4.31E+01 | 4.57E+01 | 0.44 | ||
| MCT | LAC ↔ LAC.x | 8.01E+02 | 8.01E+02 | 8.04E+02 | 6.49E+02 | 6.49E+02 | 6.49E+02 | 0.81 | ||
| ALAR | ALA → ALA.x | 1.43E+01 | 1.43E+01 | 1.46E+01 | 7.83E+00 | 7.83E+00 | 8.24E+00 | 0.55 | ||
| GLNR | GLN.x → GLN | 7.73E+01 | 7.53E+01 | 7.73E+01 | 1.77E+02 | 1.77E+02 | 1.77E+02 | 2.29 | ||
| GLUR | GLU ↔ GLU.x | 2.53E+01 | 2.52E+01 | 2.54E+01 | 1.19E+01 | 1.19E+01 | 1.22E+01 | 0.47 | ||
| ASPR | ASP → ASP.x | 7.01E+00 | 6.99E+00 | 7.02E+00 | 6.92E+00 | 6.84E+00 | 7.00E+00 | |||
| SERR | SER.x → SER | 2.54E+00 | 2.48E+00 | 2.55E+00 | 2.57E+00 | 2.55E+00 | 2.57E+00 | 1.01 | ||
| CYSR | CYX.x → CYS +CYS | 6.39E+00 | 6.34E+00 | 6.45E+00 | 3.75E+00 | 3.75E+00 | 3.75E+00 | 0.59 | ||
| GLYR | GLY → GLY.x | 3.66E-01 | 3.03E-01 | 4.19E-01 | 4.06E-01 | 3.86E-01 | 4.25E-01 | |||
| Glycolysis | HK | GLC → G6P | 4.28E+02 | 4.28E+02 | 4.28E+02 | 3.65E+02 | 3.65E+02 | 3.65E+02 | 0.85 | |
| PGI | G6P ↔ F6P | 4.06E+02 | 4.06E+02 | 4.07E+02 | 3.62E+02 | 3.62E+02 | 3.63E+02 | 0.89 | ||
| PFK | F6P → FBP | 4.17E+02 | 4.17E+02 | 4.18E+02 | 3.61E+02 | 3.60E+02 | 3.61E+02 | 0.87 | ||
| ALDO | FBP ↔ DHAP +GAP | 4.17E+02 | 4.17E+02 | 4.18E+02 | 3.61E+02 | 3.60E+02 | 3.61E+02 | 0.87 | ||
| TPI | DHAP ↔ GAP | 4.16E+02 | 4.16E+02 | 4.16E+02 | 3.60E+02 | 3.60E+02 | 3.60E+02 | 0.87 | ||
| GAPDH | GAP ↔ 3 PG | 8.39E+02 | 8.39E+02 | 8.41E+02 | 7.21E+02 | 7.21E+02 | 7.21E+02 | 0.86 | ||
| ENO | 3 PG → PEP | 8.36E+02 | 8.35E+02 | 8.53E+02 | 7.20E+02 | 7.20E+02 | 7.20E+02 | 0.86 | ||
| PK | PEP → PYR.c | 9.31E+02 | 9.30E+02 | 9.31E+02 | 9.24E+02 | 9.24E+02 | 9.24E+02 | 0.99 | ||
| LDH | PYR.c ↔ LAC | 8.01E+02 | 8.01E+02 | 8.04E+02 | 6.49E+02 | 6.49E+02 | 6.49E+02 | 0.81 | ||
| GPT1 | PYR.c ↔ ALA | 1.64E+02 | 1.62E+02 | 1.92E+02 | –1.36E+01 | –1.39E+01 | –1.35E+01 | –0.08 | ||
| GPT2 | PYR.m ↔ ALA | –1.43E+02 | –1.43E+02 | –1.42E+02 | 2.62E+01 | 2.51E+01 | 2.65E+01 | –0.18 | ||
| Pentose phosphate pathway | G6PD | G6P → P5P+CO2 | 1.89E+01 | 1.57E+01 | 1.93E+01 | 1.16E-07 | 0.00E+00 | 1.10E-03 | 0 | |
| TK1 | P5P+P5 P ↔ S7P+GAP | 5.46E+00 | 4.44E+00 | 5.96E+00 | –6.15E-01 | –6.15E-01 | –5.77E-01 | –0.11 | ||
| TA | S7P+GAP ↔ F6P+E4 P | 5.46E+00 | 4.44E+00 | 5.96E+00 | –6.15E-01 | –6.15E-01 | –5.77E-01 | –0.11 | ||
| TK2 | P5P+E4 P ↔ F6P+GAP | 5.46E+00 | 4.44E+00 | 5.96E+00 | –6.15E-01 | –6.15E-01 | –5.77E-01 | –0.11 | ||
| Anaplerosis | PYRT | PYR.c → PYR.m | 7.60E+01 | 7.59E+01 | 7.66E+01 | 3.36E+02 | 3.36E+02 | 3.36E+02 | 4.42 | |
| PC | PYR.m+CO2 → OAC | 6.30E+01 | 6.29E+01 | 6.59E+01 | 2.37E+02 | 2.36E+02 | 2.37E+02 | 3.76 | ||
| PEPCK | OAC → PEP +CO2 | 9.51E+01 | 9.51E+01 | 9.53E+01 | 2.03E+02 | 2.03E+02 | 2.04E+02 | 2.14 | ||
| ME2 | MAL → PYR.m +CO2 | 1.20E-03 | 0.00E+00 | 5.20E-03 | 1.82E+02 | 1.81E+02 | 1.82E+02 | 151517.08 | ||
| ME1 | MAL → PYR.c +CO2 | 3.29E-05 | 0.00E+00 | 1.15E+00 | 5.91E-05 | 0.00E+00 | 8.06E-02 | |||
| FAO | FAO → AcCoA.m | 1.00E-07 | 0.00E+00 | 1.32E-02 | 1.15E-04 | 0.00E+00 | 1.56E-01 | |||
| GLDH | GLU ↔ AKG | 4.43E+01 | 4.42E+01 | 4.45E+01 | 1.59E+02 | 1.59E+02 | 1.59E+02 | 3.6 | ||
| GLS | GLN ↔ GLU | 7.38E+01 | 7.36E+01 | 7.38E+01 | 1.74E+02 | 1.74E+02 | 1.74E+02 | 2.36 | ||
| Tricarboxylic acid cycle | PDH | PYR.m → AcCoA.m +CO2 | 1.56E+02 | 1.48E+02 | 1.66E+02 | 2.55E+02 | 2.55E+02 | 2.55E+02 | 1.63 | |
| CS | AcCoA.m+OAC → CIT | 1.56E+02 | 1.56E+02 | 1.58E+02 | 2.55E+02 | 2.55E+02 | 2.55E+02 | 1.63 | ||
| IDH | CIT ↔ AKG +CO2 | 2.11E+01 | 2.10E+01 | 2.11E+01 | 2.16E+01 | 2.16E+01 | 2.16E+01 | 1.03 | ||
| OGDH | AKG → SUC +CO2 | 6.54E+01 | 6.51E+01 | 6.59E+01 | 1.81E+02 | 1.80E+02 | 1.81E+02 | 2.77 | ||
| SDH | SUC ↔ FUM | 6.54E+01 | 6.51E+01 | 6.59E+01 | 1.81E+02 | 1.80E+02 | 1.81E+02 | 2.77 | ||
| FH | FUM ↔ MAL | 6.54E+01 | 6.51E+01 | 6.59E+01 | 1.81E+02 | 1.80E+02 | 1.81E+02 | 2.77 | ||
| MDH | MAL ↔ OAC | 2.01E+02 | 2.01E+02 | 2.01E+02 | 2.32E+02 | 2.32E+02 | 2.33E+02 | 1.16 | ||
| GOT | OAC ↔ ASP | 1.22E+01 | 1.17E+01 | 1.24E+01 | 1.07E+01 | 1.06E+01 | 1.07E+01 | 0.87 | ||
| Amino acid metabolism | PST | 3 PG → SER | 2.69E+00 | 2.57E+00 | 2.80E+00 | 7.12E-01 | 7.01E-01 | 7.21E-01 | 0.26 | |
| SHT | SER ↔ GLY +MEETHF | 5.19E+00 | 5.15E+00 | 5.20E+00 | 3.82E+00 | 3.81E+00 | 3.86E+00 | 0.74 | ||
| CYST | SER ↔ CYS | –1.12E+01 | –1.17E+01 | –1.11E+01 | –6.35E+00 | –6.35E+00 | –6.35E+00 | 0.57 | ||
| SD | SER → PYR.c | 6.39E+00 | 6.23E+00 | 6.44E+00 | 2.33E+00 | 2.33E+00 | 2.33E+00 | 0.36 | ||
| GLYS | CO2 +MEETHF → GLY | 2.39E+00 | 2.36E+00 | 2.42E+00 | 1.80E+00 | 1.79E+00 | 1.81E+00 | 0.75 | ||
| Biomass | BIOMASS | 978*AcCoA.c+237.8* ALA +187*ASP +92.3* CO2 +57.46*CYS +45.97*DHAP +114.5*G6P+127.6* GLN +153*GLU +260.8*GLY +101.1*MEETHF +92.3*P5P+174.8* SER → biomass | 2.77E-02 | 2.70E-02 | 2.79E-02 | 2.00E-02 | 2.00E-02 | 2.00E-02 | 0.72 | |
| ACL | CIT → AcCoA.c +MAL | 1.35E+02 | 1.34E+02 | 1.38E+02 | 2.33E+02 | 2.33E+02 | 2.33E+02 | 1.72 | ||
| LIPS | AcCoA.c → lipid | 1.08E+02 | 9.99E+01 | 1.08E+02 | 2.14E+02 | 2.14E+02 | 2.14E+02 | 1.98 | ||
| Mixing | cPYR | 0*PYR.c → PYR.ms | 5.77E-01 | 5.64E-01 | 5.92E-01 | 1.00E+00 | 9.96E-01 | 1.00E+00 | 1.73 | |
| mPYR | 0*PYR.m → PYR.ms | 4.23E-01 | 4.08E-01 | 4.36E-01 | 1.00E-07 | 0.00E+00 | 4.40E-03 | 0 | ||
| sPYR | PYR.ms → PYR.fix | 1.00E+00 | 1.00E+00 | 1.00E+00 | 1.00E+00 | 1.00E+00 | 1.00E+00 | |||
| EXCH | Transport | MCT | LAC ↔ LAC.x | 1.00E-07 | 0.00E+00 | 1.36E+02 | 1.64E+03 | 1.63E+03 | 1.65E+03 | 16400000000 |
| GLUR | GLU ↔ GLU.x | 1.00E-07 | 0.00E+00 | 2.27E-02 | 5.69E-05 | 0.00E+00 | 1.71E-02 | |||
| Glycolysis | PGI | G6P ↔ F6P | 4.88E+06 | 4.88E+06 | Inf | 9.92E+06 | 9.85E+04 | Inf | ||
| ALDO | FBP ↔ DHAP +GAP | 2.89E+02 | 2.80E+02 | 2.89E+02 | 2.57E+02 | 2.56E+02 | 2.57E+02 | 0.89 | ||
| TPI | DHAP ↔ GAP | 9.86E+06 | -Inf | Inf | 1.65E+03 | 1.63E+03 | 1.68E+03 | |||
| GAPDH | GAP ↔ 3 PG | 1.12E+03 | 0.00E+00 | 5.88E+05 | 1.00E-07 | 0.00E+00 | 2.27E-01 | |||
| LDH | PYR.c ↔ LAC | 1.47E+03 | 1.39E+03 | 1.47E+03 | 4.49E+02 | 4.49E+02 | 4.49E+02 | 0.31 | ||
| GPT1 | PYR.c ↔ ALA | 2.74E+02 | 2.73E+02 | 2.77E+02 | 1.00E-07 | 0.00E+00 | 4.28E-02 | 0 | ||
| GPT2 | PYR.m ↔ ALA | 1.38E+02 | 1.38E+02 | 1.49E+02 | 9.64E+01 | 0.00E+00 | 1.01E+02 | 0.7 | ||
| Pentose phosphate pathway | TK1 | P5P+P5 P ↔ S7P+GAP | 7.99E+02 | 7.97E+02 | 8.08E+02 | 3.54E+01 | 3.54E+01 | 3.55E+01 | 0.04 | |
| TA | S7P+GAP ↔ F6P+E4 P | 1.53E-01 | 0.00E+00 | 5.82E-01 | 2.55E+00 | 2.54E+00 | 2.57E+00 | 16.67 | ||
| TK2 | P5P+E4 P ↔ F6P+GAP | 3.33E+00 | 2.62E+00 | 3.35E+00 | 1.29E+01 | 1.29E+01 | 1.29E+01 | 3.88 | ||
| Anaplerosis | GLDH | GLU ↔ AKG | 5.36E+02 | 5.34E+02 | 8.37E+02 | 1.23E+03 | 1.23E+03 | 1.23E+03 | 2.29 | |
| GLS | GLN ↔ GLU | 3.20E-01 | 0.00E+00 | 2.74E+00 | 1.12E+00 | 1.07E+00 | 1.74E+00 | |||
| Tricarboxylic acid cycle | IDH | CIT ↔ AKG +CO2 | 1.04E+01 | 1.02E+01 | 1.04E+01 | 6.30E+01 | 6.30E+01 | 6.31E+01 | 6.09 | |
| SDH | SUC ↔ FUM | 2.78E-01 | 0.00E+00 | Inf | 3.34E+06 | 3.34E+06 | 3.34E+06 | |||
| FH | FUM ↔ MAL | 1.03E-04 | 0.00E+00 | 1.58E+01 | 2.18E+02 | 2.18E+02 | 2.18E+02 | 2114238.83 | ||
| MDH | MAL ↔ OAC | 1.01E+03 | 8.27E+02 | 1.01E+03 | 3.67E+03 | 3.67E+03 | 3.69E+03 | 3.63 | ||
| GOT | OAC ↔ ASP | 2.27E+02 | 2.27E+02 | 2.47E+02 | 1.54E+01 | 1.54E+01 | 1.55E+01 | 0.07 | ||
| Amino acid metabolism | SHT | SER ↔ GLY +MEETHF | 3.55E+00 | 3.52E+00 | 3.59E+00 | 1.60E-01 | 1.36E-01 | 1.70E-01 | 0.05 | |
| CYST | SER ↔ CYS | 1.04E+03 | 1.03E+03 | 1.04E+03 | 2.00E-03 | 0.00E+00 | 2.00E-03 | 0 | ||
-
*
SSR 575.6 [499.1–630.6] (95% CI, 563 DOF).
-
†
SSR 521.3 [482.2–611.6] (95% CI, 545 DOF).
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | anti-HIF-1α (Mouse monoclonal) | BD Biosciences | 610958 | 1:1000 |
| Antibody | anti-c-MYC (Rabbit monoclonal) | Cell Signaling Technologies | D84C12 | 1:1000 |
| Antibody | anti-LDHA (Rabbit polyclonal) | Cell Signaling Technologies | 2012 | 1:1000 |
| Antibody | HRP-anti-Rabbit IgG (Goat polyclonal) | Cell Signaling Technologies | 7074 | 1:5000 |
| Antibody | HRP-anti-Mouse IgG (Goat polyclonal) | Cell Signaling Technologies | 7076 | 1:5000 |
| Transfected construct (human) | MYC | Vector Biolabs | 1285 | adenovirus to express MYC |
| Transfected construct (human) | YFP | Oldham et al., 2015 | adenovirus control to express YFP | |
| Chemical compound, drug | [1,2–13 C2]-glucose | Cambridge Isotope Labs | CLM-504-PK | |
| Chemical compound, drug | [U-13C6]-glucose | Cambridge Isotope Labs | CLM-1396-PK | |
| Chemical compound, drug | [U-13C5]-glutamine | Cambridge Isotope Labs | CLM-1822-H-PK | |
| Chemical compound, drug | [U-13C3]-lactate | Sigma | 485926 | |
| Chemical compound, drug | BAY | Cayman | 15297 | Molidustat (BAY-85–3934); 10 μM in DMSO |
| Commercial assay or kit | Glucose colorimetric assay kit | Cayman | 10009582 | |
| Commercial assay or kit | ʟ-Lactate assay kit | Cayman | 700510 | |
| Commercial assay or kit | Pyruvate assay kit | Cayman | 700470 | |
| Commercial assay or kit | NADP/NADPH-Glo Assay | Promega | G9081 | |
| Cell line (human) | LFs | Lonza | CC-2512 | Normal human lung fibroblasts |
| Cell line (human) | PASMCs | Lonza | CC-2581 | Pulmonary artery smooth muscle cells |
| Sequence-based reagent | ACTB | Life Technologies | Hs03023943_g1 | qPCR probe |
| Sequence-based reagent | GLUT1 | Life Technologies | Hs00892681_m1 | qPCR probe |
| Sequence-based reagent | LDHA | Life Technologies | Hs00855332_g1 | qPCR probe |
| Sequence-based reagent | CDKN1A | Life Technologies | Hs00355782_m1 | qPCR probe |
| Transfected construct (human) | siMYC | Dharmacon | L-003282-02-0005 | ON-TARGETplus siRNA |
| Transfected construct (human) | siPHD2 | Dharmacon | L-004276-00-0005 | ON-TARGETplus siRNA |
| Transfected construct (human) | siCTL | Dharmacon | D-001810-10-05 | ON-TARGETplus non-targeting control pool |
