Figure 2

Effects of digitoflavone on mRNA and protein levels of Nrf2-mediated phase II enzymes-antioxidant proteins, Nrf2 expression and nucleus accumulation in Caco-2 cells. (A) Effects of digitoflavone on mRNA levels of the selected ARE genes. Caco-2 cells were treated with 10 μM digitoflavone for 8 hours and the mRNA levels of all selected genes were evaluated using RT-PCR. (B) Dose and time effect of digitoflavone. Caco-2 cells were treated with various concentrations (0–15 μM) of digitoflavone for 8 hours (top) and 10 μM digitoflavone for 0–8 hours (bottom). Nrf2, γ-GCSc, γ-GCSm and TR expression level were determined using Western blotting. (C) Time effect of Nrf2 nucleus accumulation. Caco-2 cells were treated with 10 μM digitoflavone for 0–8 hours. Total form Nrf2 in nucleus (N) and cytoplasm (C) were determined using Western blotting. (D) Immunofluorescence staining of Nrf2. Caco-2 cells were treated with 10 μM digitoflavone for 0–8 hours and then Nrf2 was labeled with immunofluorescence staining. Cells were counter-stained with DAPI for visualization of the nuclei. The left panel shows the relative signal-intensity profiles of Nrf2 and DAPI along the cross sections at the light yellow line. White scale bar indicates 25 μm. (E) Nrf2 silencing abrogates digitoflavone-induced cytoprotective protein expression. After 48 h transfection, the cells were treated with 10 μM digitoflavone for 8 hours and whole-cell lysates (TL) were then prepared. The protein levels of Nrf2, γ-GCSc, γ-GCSm and TR were analyzed. (F-H) Effects of digitoflavone on cellular total GSH, GSSG and the ratio of GSH/GSSG. Caco-2 cells were treated with various concentrations of digitoflavone for 8 hours, and the total GSH, GSSG and the GSH/GSSG ratio were analyzed according to the manufacturer’s instructions. The values represent the means ± SD of triplicate experiments. Significantly different (versus control group): *p<0.05, **p<0.01.