Fig. 2: Involvement of lysosomes 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 at the indicated concentration for 24 h. Then, immunostaining was performed using LAMP2 and SeP antibodies. Fluorescence was observed with a confocal microscope (a). The scale bar indicates 20 µm. HepG2 cells were cultured in high glucose DMEM containing selenite (100 nM) for 24 h and treated with SFN at the indicated concentration for 24 h. After that, the cells were treated with Lysotracker, and fluorescence was observed with a confocal microscope (b). The scale bar indicates 20 µm. The cells were treated with SFN at the indicated concentration for 24 h. ATP6V1A mRNA was measured using RT-qPCR (c). HepG2 cells were cultured in high glucose DMEM containing selenite (100 nM) for 24 h and treated with 6 µM SFN in the presence of the indicated concentration of chloroquine (CQ) (d) or bafilomycin A1 (BafA1) (e) for 24 h. The culture medium supernatant and cell lysate were collected, and western blotting was performed. Quantitative data is shown at the lower panel of each figure and indicated as a relative value with the control as 1. The data is expressed as mean ± S.D. (n = 3). *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.