FIGURE 2.

Nrf2-dependent induction of proteasome activity in NCM460 cells by coculture with inflammatory macrophages. A and B, NCM460 cells either monocultured (mono) or cocultured (3 days) with macrophages (M-g and M-m) were submitted to the Suc-LLVY-AMC assay (A) or NCM460 cells treated with control, Nrf1, or Nrf2 siRNA and cultured alone (mono) or with GM-CSF macrophages (M-g) were submitted to the Suc-LLVY-AMC assay (B, upper panel) or real time RT-PCR (B, lower panels). Specific proteasome activity (A and B, upper panel) was determined by the release of fluorogenic AMC normalized to the protein content; the data represent the means ± S.D. of six independent experiments. *, p < 0.05. Real time RT-PCR was conducted using Nrf1, Nrf2, and β-actin primers (B, lower panels), and data from three independent experiments (means ± S.D.) performed in duplicate are shown. C and D, NCM460 cells treated with control, Nrf1, or Nrf2 siRNA and cultured (3 days) alone (mono) or with G-MCSF macrophages (M-g) were submitted to real time PCR using S5a, α5, and β-actin primers (C) or to immunoblotting using S5a, α5, and Hsp90 antibodies (D). Data from three independent experiments (means ± S.D.) performed in duplicate are shown in C, and D shows representative blots and the normalized S5a and α5 band intensities as determined by band densitometry of blots from three independent experiments (means ± S.D.).