Fig. 3. Involvement of Nrf2 activation in the reduction of SeP by SFN.
HepG2 cells were cultured in high glucose DMEM containing selenite (100 nM) for 24 h and treated with SFN. SFN concentration-dependency of Nrf2 activation for 24 h (a), and HO-1 mRNA expression level (b) or time-dependency of Nrf2 activation with SFN at 6 µM (c) and HO-1 levels (d) are shown. Control and Nrf2 siRNA were transfected into HepG2 cells and cultured for 24 h. The cells were treated with SFN at the indicated concentrations for 24 h. Cell lysate and culture supernatant were collected, and western blotting was performed (e). Representative blotting (left panel) and quantifications of the band intensity (right panel, n = 3) are shown. WT, Nrf2 KO, Keap1flox/flox::Alb-Cre mouse serum was collected and SeP was detected by western blotting (f, quantifications of the band intensity are shown in the right panel (WT n = 6, Nrf2 KO n = 3, and Keap1flox/flox::Alb-Cre n = 5). Control and Nrf2 siRNA were introduced into HepG2, cultured for 24 h, and treated with SFN at the indicated concentrations for 24 h. After that, the cells were stained with Lysotracker and observed with a confocal microscope (g). The scale bar indicates 20 µm. Under the same conditions, ATP6V1A mRNA was measured by RT-qPCR (h). The scheme of the underlying mechanism suppressing SeP expression by SFN is shown (i). The data is expressed as mean ± S.D. (n = 3) and indicated as a relative value with the control as 1. *P < 0.05 and **P < 0.01 vs. control. One-way ANOVA, post hoc test Dunnett method were used for statistical analysis. All blots were performed on independent membranes and were done with the same sample volume applied. CBB staining was performed on the same membrane.