FIGURE 4.

The Nrf2-induced proteasome activity in NCM460 cells by inflammatory macrophages depends on ROS. A, NCM460 cells were either monocultured (mono) or cocultured with G-MCSF macrophages (M-g) for 3 days in the absence or presence of 0.2 mm Tiron or 200 units of catalase, or NCM460 cells were cocultured with G-MCSF macrophages pretreated (pre) with Tiron or catalase for 16 h. Upon either treatment, NCM460 cells were submitted to the Suc-LLVY-AMC assay. The data represent the means ± S.D. of four independent experiments performed in duplicate. *, p < 0.05. B, NCM460 cells either monocultured (mono) or cocultured (M-g) for 3 days in the absence or presence of Tiron or catalase or cocultured with Tiron or catalase pretreated (pre) GM-CSF macrophages (M-g) were transfected with pARE or the control (pGL3) firefly luciferase reporter vector together with ptkRL. After further culture for 24 h, NCM460 cell lysates were analyzed for firefly and Renilla luciferase expression. The data indicate the n-fold ARE-specific firefly luciferase (upon normalization to Renilla luciferase) and represent the means ± S.D. of four independent experiments performed in duplicate. *, p < 0.05. C and D, NCM460 cells cultured either without (mono) or with GM-CSF macrophages (M-g) for 3 days in the absence or presence of Tiron were analyzed for the expression of S5a and α5 by real time PCR (D) and Western blot (E). Data from three independent experiments (means ± S.D.) performed in duplicate are shown in C, and D shows a representative blot and the normalized S5a and α5 band intensities as determined by band densitometry of blots from three independent experiments (means ± S.D.).