Effect of TRAF6 gene silencing on cytokine production by E coli–treated and adenosine-stimulated macrophages. (A) TRAF6 mRNA level was decreased in RAW 264.7 macrophages stably transfected with TRAF6 shRNA compared with scrambled shRNA. TRAF6 mRNA level was measured using real-time reverse-transcription polymerase chain reaction using total RNA isolated from untransfected, scrambled shRNA-transfected and TRAF6 shRNA-transfected RAW 264.7 macrophages. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 4 in each experiment. ^###P < .001. (B) IL-6 production was decreased by TRAF6 gene silencing and the suppressive effect of adenosine is TRAF6-dependent in E coli-stimulated macrophages. RAW 264.7 cells were transfected with a plasmid generating TRAF6 shRNAs for silencing of TRAF6. The transfected macrophages were treated with heat-killed E coli or E coli plus 100 μM adenosine for 5 hours, and IL-6 levels were measured using enzyme-linked immunosorbent assay from the supernatants taken at the end of the incubation period. ^###P < .001 vs. scrambled. (C) Effect of TRAF6 silencing on IL-10 production by macrophages exposed to E coli or E coli plus adenosine. IL-10 production was determined from the supernatants that were obtained from stably transfected RAW 264.7 cells after 5 hours of exposure to E coli or E coli plus adenosine. *P < .05 vs. scrambled. ***P < .001 vs. scrambled. Results (mean ± SEM) shown are representative of at least 3 experiments with n = 4 in each experiment.