American Society of Hematology

Figure 6

$Figure 6. Adenosine enhances C/EBPβ activation in E coli–stimulated macrophages. (A) Adenosine up-regulates C/EBP–luciferase activity in macrophages exposed to E coli. RAW 264.7 macrophages were transfected with a luciferase reporter vector driven by C/EBP (pC/EBP-luc) or a control vector (pCIS-CK). Cells were treated with E coli and adenosine (100 μM) for 8 hours, after which period the cells were lysed and luciferase activity determined. Luciferase reporter activities were normalized to protein concentration. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 3 in each experiment. *P < .05 and **P < .01 vs. control. (B) Adenosine enhances E coli–induced C/EBPβ DNA binding. RAW cells were treated with heat-killed E coli or simultaneously with 100 μM adenosine and heat-killed E. coli, and nuclear proteins were extracted at 30, 60, and 90 minutes thereafter. C/EBPβ DNA binding of nuclear proteins was measured using electrophoretic mobility shift assay. The upper panel shows densitometric analysis of intensities of complexes (fold increase vs. control) observed on the gel (lower panel). (C) An antibody against C/EBPβ shifts the adenosine/E coli–induced (for 60 minutes) DNA–protein complex. For supershift studies nuclear extracts were preincubated with C/EBPβ and C/EBPδ antibodies before the binding reaction, and the complexes were separated by electrophoretic mobility shift assay and visualized using autoradiography. The figure shown is representative of 3 separate experiments. (Di) Adenosine increases E coli–induced C/EBPβ protein accumulation in the nuclear fraction of RAW 264.7 cells. Cytosolic and nuclear protein extracts were taken at 30 and 60 minutes after exposure to E coli or E coli plus adenosine (100 μM) and C/EBPβ expression was determined by Western blotting and autoradiography. (Dii)Adenosine alone fails to affect C/EBPβ protein accumulation in the nuclear fraction of RAW 264.7 cells. The figures shown are representative of 3 separate experiments. (E) Densitometric analysis of the 3 C/EBPβ isoforms detected by autoradiography on the blot shown in (Di; fold increase vs. control). The figure is representative of 3 separate experiments. (F) E coli both in the absence and presence of adenosine fails to increase IL-10 production by C/EBPβ-deficient macrophages. C/EBPβ WT and KO macrophages were challenged with E coli or E coli plus adenosine and IL-10 production was determined from the supernatants after a 5-hour stimulation. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 6 in each experiment. ***P < .001 vs. E coli.$

Adenosine enhances C/EBPβ activation in E coli–stimulated macrophages. (A) Adenosine up-regulates C/EBP–luciferase activity in macrophages exposed to E coli. RAW 264.7 macrophages were transfected with a luciferase reporter vector driven by C/EBP (pC/EBP-luc) or a control vector (pCIS-CK). Cells were treated with E coli and adenosine (100 μM) for 8 hours, after which period the cells were lysed and luciferase activity determined. Luciferase reporter activities were normalized to protein concentration. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 3 in each experiment. *P < .05 and **P < .01 vs. control. (B) Adenosine enhances E coli–induced C/EBPβ DNA binding. RAW cells were treated with heat-killed E coli or simultaneously with 100 μM adenosine and heat-killed E. coli, and nuclear proteins were extracted at 30, 60, and 90 minutes thereafter. C/EBPβ DNA binding of nuclear proteins was measured using electrophoretic mobility shift assay. The upper panel shows densitometric analysis of intensities of complexes (fold increase vs. control) observed on the gel (lower panel). (C) An antibody against C/EBPβ shifts the adenosine/E coli–induced (for 60 minutes) DNA–protein complex. For supershift studies nuclear extracts were preincubated with C/EBPβ and C/EBPδ antibodies before the binding reaction, and the complexes were separated by electrophoretic mobility shift assay and visualized using autoradiography. The figure shown is representative of 3 separate experiments. (Di) Adenosine increases E coli–induced C/EBPβ protein accumulation in the nuclear fraction of RAW 264.7 cells. Cytosolic and nuclear protein extracts were taken at 30 and 60 minutes after exposure to E coli or E coli plus adenosine (100 μM) and C/EBPβ expression was determined by Western blotting and autoradiography. (Dii)Adenosine alone fails to affect C/EBPβ protein accumulation in the nuclear fraction of RAW 264.7 cells. The figures shown are representative of 3 separate experiments. (E) Densitometric analysis of the 3 C/EBPβ isoforms detected by autoradiography on the blot shown in (Di; fold increase vs. control). The figure is representative of 3 separate experiments. (F) E coli both in the absence and presence of adenosine fails to increase IL-10 production by C/EBPβ-deficient macrophages. C/EBPβ WT and KO macrophages were challenged with E coli or E coli plus adenosine and IL-10 production was determined from the supernatants after a 5-hour stimulation. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 6 in each experiment. ***P < .001 vs. E coli.

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