Fig. 3.

FXR agonist GW4064 mitigates cisplatin-induced ferroptotic responses in HK2 cells.

(A) HK2 cells were transfected with siControl or siFXR as indicated; 48 h later, the cells were treated with cisplatin (10 μM) for 24 h. The levels of MDA and iron, and GSH/GSSG ratio in HK2 cells (n = 6). (B) Protein levels of GPX4, HMOX1, and FXR were detected by immunoblotting. The relative protein levels are shown. The values for vehicle-treated siControl group were set to 1 (n = 3). (C–E) After treatment with GW4064 (1 μM) for 2 h, HK2 cells were treated with cisplatin (10 μM) for another 24 h. (C) Protein levels of GPX4 and HMOX1 were detected by immunoblotting. The relative protein levels are shown. The values for the vehicle group were set to 1 (n = 4). (D) The relative levels of MDA and iron as well as GSH/GSSG ratio are shown. (E) After treatment with cisplatin and GW4064, the cells were incubated with C11-BODIPY 581/591 (2 μM) and Hoechst 33258 for counterstaining; the images were then immediately visualized by confocal microscopy. Confocal microscopy showed non-oxidized lipid (red, Texas Red) and oxidized lipid (green, FITC). scale bar, 50 μm. All values are presented as the mean ± SD. Statistical significance was measured using one- or two-way ANOVA with Bonferroni post hoc-test. *P < 0.05, **P < 0.005. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)