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. 2020 Jan 20;10:670. doi: 10.1038/s41598-019-57368-w

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© The Author(s) 2020

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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Importance of glycolysis for LPS-induced Pdgfb mRNA expression in macrophages. (a) Schematic pathway of glycolytic metabolism. The terminals of half lines represent the targets of inhibitors. (b) Relative expression levels of Pdgfb mRNA in peritoneal macrophages pretreated with 10 mM 2-DG for 0.5 hours and stimulated with 100 ng/mL LPS or 10 ng/mL IL-4 for 24 hours under 11 mM glucose. n = 4. (c–f) Relative expression levels of Pdgfb, Tnfa, and Il6 mRNA in RAW264.7 cells pretreated with 2-DG or HA for 0.5 hours and stimulated with LPS for 3 hours under 11 mM glucose. Mean of data among three or four independent experiments. n = 6 or 5. (g) Representative images blotted with anti-GAPDH and anti-α-Tubulin antibodies, and relative signal density of GAPDH normalized with that of α-Tubulin in siGapdh-transfected RAW264.7 cells. Mean of data among three independent experiments. n = 6. (h) Relative expression levels of Pdgfb mRNA in RAW264.7 cells stimulated with LPS for 3 hours under 11 mM glucose after siGapdh transfection. Mean of data among three independent experiments. n = 6. (i) Relative expression levels of Pdgfb mRNA in RAW264.7 cells pretreated with 10 mM 2-DG for 0.5 hours, and stimulated with LPS for 3 hours under 11 mM glucose with the following co-treatment, as indicated: 20 mM pyruvate, 20 mM lactic acid (LA), 20 mM LA neutralized by 16.1 mM NaOH, or culture medium adjusted with 18.8 mM HCl. Mean of data among three independent experiments. n = 6. (j–l) Relative expression levels of Pdgfb, Tnfa, and Il6 mRNA in RAW264.7 cells stimulated with LPS in the presence or absence of 20 mM LA for 3 hours under 11 mM glucose. Mean of data among five independent experiments. n = 5. Data are shown as means ± S.E. *p < 0.05 and **p < 0.01, among two groups, as indicated. HK, hexokinase; G6P, glucose-6-phosphate; GAP, glyceraldehyde 3-phosphate; GAPDH, glyceraldehyde phosphate dehydrogenase; 1,3-BPG, D-glycerate 1,3-bisphosphate; 2-DG, 2-deoxyglucose; HA, heptelidic acid.