Figure 5

Relevance of p65 and mTORC1 signaling pathways to LPS-induced Pdgfb mRNA expression coupled with glycolysis in macrophages. (a) Representative images blotted with anti-phospho-p65 and anti-p65 antibodies, and relative signal density of phospho-p65 normalized with that of p65 in RAW264.7 cells pretreated with 10 mM 2-DG for 0.5 hour and stimulated with 100 ng/mL LPS for 0.5 hours. Mean of data among four independent experiments. n = 4. Statistical analyses were performed under the same glucose concentration. (b) Representative images blotted with anti-phospho-p65 and anti-p65 antibodies, and relative signal density of phospho-p65 normalized with that of p65 in RAW264.7 cells pretreated with HA for 0.5 hours and stimulated with 100 ng/mL LPS for 3 hours. Mean of data among three independent experiments. n = 4. (c) Representative images blotted with anti-p65 and anti-α-Tubulin antibodies, and relative signal density of p65 normalized with that of α-Tubulin in siRela-transfected RAW264.7 cells. Mean of data among three independent experiments. n = 6. (d,e) Relative expression levels of Il6 and Pdgfb mRNA in RAW264.7 cells stimulated with 100 ng/mL LPS for 3 hours after siRela transfection. Mean of data among three independent experiments. n = 6. (f) Representative images blotted with anti-phospho-S6K and anti-S6K antibodies, and relative signal density of phospho-S6K normalized with that of S6K in RAW264.7 cells pretreated with 10 mM 2-DG for 0.5 hours and stimulated with 100 ng/mL LPS for 3 hours. Mean of data among four independent experiments. n = 4. Statistical analyses were performed under the same glucose concentration. (g,h) Relative expression levels of Il6 and Pdgfb mRNA in RAW264.7 cells pretreated with 10 nM rapamycin (Rapa) for 1 hour and stimulated with 100 ng/mL LPS for 3 hours. Mean of data among four independent experiments. n = 8. Data are shown as means ± S.E. *p < 0.05 and **p < 0.01, among two groups, as indicated. A.U., arbitrary unit.