FIGURE 6.
Activation of the A2A receptor prevents LPS-IFNγ-induced and HIF-1-mediated TfR1 expression by inhibiting NF-κB activity. A, RLA in RAW 264.7 cells untreated (C) or treated as described in Fig. 5A. The cells were transiently transfected with the empty pTAL vector (ev) or a construct in which luciferase was controlled by a NF-κB multimer and co-transfected with the Renilla luciferase vector. Luciferase activity was determined after 24 h and calculated as described in Fig. 2C.*, p < 0.001 versus untreated controls; **, p < 0.001 versus cells treated with LPS/IFNγ. B, RLA in RAW 264.7 cells untreated (C) or treated with LPS-IFNγ for 4 h in the presence or absence of the NF-κB inhibitor Bay 11-7082 (BAY) (added 2 h before treatment). The cells were transiently transfected with a construct in which luciferase was controlled by a NF-κB multimer and co-transfected with the Renilla luciferase vector. Luciferase activity was determined as described above. *, p < 0.001 versus untreated controls; **, p < 0.001 versus cells treated with LPS/IFNγ. C, immunoblot analysis of cytosolic extracts from RAW 264.7 cells untreated or treated with LPS-IFNγ for 4 h in the presence or absence of BAY (added 2 h before treatment) using the antibody against TfR1. The blots were reprobed with the antibody against β-actin as a loading control. D, immunoblot analysis of nuclear extracts from RAW 264.7 cells untreated or treated as in panel C using the antibody against HIF-1α. The blots were reprobed with the antibody against TFIID as a loading control. E, immunoblot analysis of nuclear extracts from RAW 264.7 cells untreated or treated with LPS-IFNγ for 4 h in the presence or absence of chetomin (added 2 h before treatment) using the antibody against p65. The blots were reprobed with the antibody against TFIID as a loading control. F, RLA in RAW 264.7 cells untreated (C) or treated with LPS-IFNγ for 4 h in the presence or absence of chetomin (added 2 h before treatment). The cells were transiently transfected with a construct in which luciferase was controlled by a NF-κB multimer and co-transfected with the Renilla luciferase vector. Luciferase activity was determined as described above. *, p < 0.001 versus untreated control. All of the results are representative of at least three independent experiments. The values indicate the -fold difference ±S.D. in relation to the untreated control.