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. 2021 Jun 12;40(27):4538–4551. doi: 10.1038/s41388-021-01886-3

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — A Respiration rates. Left: Seahorse XF Cell Mito Stress Test performed on primary MEFs of the indicated genotypes. 20,000 cells/well were seeded and incubated for 24 h. OCR (oxygen consumption rate) was measured under basal conditions followed by the sequential addition of 1.5 µM Oligomycin (OL), 1 µM FCCP, and 1 µM Rotenone and Antimycin A (ROT/AA) following manufacturer’s instruction. Right: quantitation of parameters for basal respiration and spare respiratory capacity. Data presented are the mean ± SEM from six independent experiments using at least five technical replicates per experiment per condition. Statistics: * vs WT; ^& vs SOS1-KO; ^# vs SOS2-KO; ***^,^###p < 0.001; **^,&&^,^##p < 0.01; *^,^&p < 0.05 (n = 6). B Glycolytic rates. Left: Seahorse XF glycolytic rate assay performed on primary MEFs of the indicated genotypes. 20,000 cells/well were seeded and incubated for 24 h. ECAR (extracellular acidification rate) was measured under basal conditions followed by the sequential addition of 1 µM Rotenone and Antimycin A (ROT/AA), and 100 mM 2-deoxyglucose (2-DG) following the manufacturer’s instructions. Right: quantitation of glycolytic proton efflux rate (glycoPER) and individual parameters for basal glycolysis and compensatory glycolysis. Data expressed as the mean ± SEM compiled from five independent experiments using at least five technical replicates per experiment per condition. Statistics: * vs WT; ^# vs SOS2-KO; **p < 0.01; ^#p < 0.05 (n = 5). C ATP production rates. Left: Seahorse XF real-time ATP rate assays of primary MEFs of the indicated genotypes. Rates of mitochondrial ATP production (mitoATP, gray bars) and glycolytic ATP production (glycoATP, black bars) quantitated following manufacturer’s instruction. Right: energetic map of the four genotypes tested charting mitochondrial ATP (mitoATP) versus glycolysis-generated ATP (glycoATP). Data shown are compiled from five independent experiments using at least five technical replicates per experiment per condition. Statistics: * vs WT; ^# vs SOS2-KO; ***p < 0.001; **^,^##p < 0.01; *^,^#p < 0.05 (n = 5). D Intracellular ATP and cAMP levels. Left: measurements of total ATP content (normalized by cell number) in cellular lysates. Right: intracellular cAMP levels measurements (normalized by μg protein) in total cellular lysates. Data shown as the mean ± SEM compiled from eight independent experiments using at least three technical replicates per experiment per genotype. Statistics: * vs WT; ^# vs SOS2-KO; *^,^#p < 0.05 (n = 8).