FIG. 7.

Inactivation of AMPK relieved effect of BBR on Nrf2 activation in LPS-induced macrophages, while inactivation of Nrf2 did not affect BBR-triggered activation of AMPK. (A) RAW264.7 cells with pGFP-Nrf2 transfection were treated as described in Fig. 6. Subcellular localization of GFP-Nrf2 protein in living cells was monitored under a fluorescent microscope. (B) Quantitative analysis of GFP-Nrf2 nuclear distribution in transfected cells as described in Figure 4D. (C and D) RAW264.7 cells with nonactivity AMPKα1 (pDN-AMPKα1) transfection were treated as in A, mRNA abundance of Nrf2 target genes (C) and Nrf2 protein distribution in nucleus (D) were analyzed by qRT-PCR and Western blot, respectively. (E) RAW264.7 cells transfected with pDN-AMPKα1 (upper panel) or pDN-Nrf2 (lower panel) were treated by LPS and BBR, followed by the mRNA abundance of an AMPK target gene, CPT-1, was measured by qRT-PCR. Results are means±SEM from three independent experiments. **p<0.01. (F) Peritoneal macrophages from wild type mice (Nrf2^+/+) or Nrf2 deficient mice (Nrf2^−/−) were treated in vitro by LPS and BBR, phosphorylated-AMPKα1 (Thr172) and total AMPKα1 abundance in whole cell lysates were probed by specifical antibodies through Western blot. Representative results from three independent experiments are shown. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars