Transfection:Article Title: MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia
Article Snippet: .. DOI: https://doi.org/10.7554/eLife.82597 27 of 39 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 .. Primary normal human lung fibroblasts (LFs) were purchased from Lonza (CC- 2512) and cultured in FGM- 2 (Lonza CC- 3132).
Construct:Article Title: MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia
Article Snippet: .. DOI: https://doi.org/10.7554/eLife.82597 27 of 39 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 .. Primary normal human lung fibroblasts (LFs) were purchased from Lonza (CC- 2512) and cultured in FGM- 2 (Lonza CC- 3132).
Control:Article Title: MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia
Article Snippet: .. DOI: https://doi.org/10.7554/eLife.82597 27 of 39 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 .. Primary normal human lung fibroblasts (LFs) were purchased from Lonza (CC- 2512) and cultured in FGM- 2 (Lonza CC- 3132).
Colorimetric Assay:Article Title: MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia
Article Snippet: .. DOI: https://doi.org/10.7554/eLife.82597 27 of 39 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 .. Primary normal human lung fibroblasts (LFs) were purchased from Lonza (CC- 2512) and cultured in FGM- 2 (Lonza CC- 3132).
Pyruvate Assay:Article Title: MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia
Article Snippet: .. DOI: https://doi.org/10.7554/eLife.82597 27 of 39 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 .. Primary normal human lung fibroblasts (LFs) were purchased from Lonza (CC- 2512) and cultured in FGM- 2 (Lonza CC- 3132).
Glo Assay:Article Title: MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia
Article Snippet: .. DOI: https://doi.org/10.7554/eLife.82597 27 of 39 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 .. Primary normal human lung fibroblasts (LFs) were purchased from Lonza (CC- 2512) and cultured in FGM- 2 (Lonza CC- 3132).
Sequencing:Article Title: MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia
Article Snippet: .. DOI: https://doi.org/10.7554/eLife.82597 27 of 39 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 .. Primary normal human lung fibroblasts (LFs) were purchased from Lonza (CC- 2512) and cultured in FGM- 2 (Lonza CC- 3132).
Real-time Polymerase Chain Reaction:Article Title: MYC overrides HIF-1α to regulate proliferating primary cell metabolism in hypoxia
Article Snippet: .. DOI: https://doi.org/10.7554/eLife.82597 27 of 39 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 .. Primary normal human lung fibroblasts (LFs) were purchased from Lonza (CC- 2512) and cultured in FGM- 2 (Lonza CC- 3132).
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