FXR-agonist activated Nrf2 and induced the expressions of Nrf2 & anti-oxidant molecules in AML12 cells. a Nrf2 in cytosols was translocated into nuclei by GW4064 treatment (1.0 μM) in AML12 cells. Nrf2 was not stained in nuclei without GW4064 (arrowheads), but clearly stained in nuclei 8 h after GW4064 administration (arrows). 4′ 6-diamidino-2-phenylindole dihydrochloride (DAPI) was used for nuclear stain in blue (pseudo-colored red). Scale bar, 50 μm b The protein expression of Nrf2 and the antioxidant molecules (catalase, heme oxygenase-1; HO-1, manganese-dependent superoxide dismutase; MnSOD, and thioredoxin; TRX) 36 h after the treatment with GW4064 (1.0 μM). c GW4064 induced phosphorylation of Akt and anti-apoptotic molecules (Bcl-xL and Bcl-2), and suppressed the expressions of harmful hepatic molecules such as FasL and Fas in AML12 liver cells. d Ablation of GW4064 by p62/SQSTM1 siRNA reduced phosphorylation of Akt, which was induced by GW4064 in AML12 liver cells, though this does not affect the protein expressions of the other molecules. e GW4064 did not affect cell cycle-associated molecules. GW4064 did not phosphorylate STAT3 (Y705) nor induce cyclinD1 and PCNA. Each experiment was performed three times and representative photographs are shown. The duplicates of immunoblots are taken from the specimens of experiments performed at different times. ImageJ software was used for quantitative analysis of western blot. The quantitative analysis data are expressed as mean ± SEM.
p values <0.05 were considered statistically significant (*: p < 0.05 vs GW-0 μM group; **: p < 0.05 vs GAPDH siRNA & GW-1.0 μM group)