FIGURE 1.

ACR induced ferroptosis in Sertoli cells. (A) Effect of ACR on intracellular GSH level. TM4 cells were cultured with the indicated concentrations of ACR for 3 h. The cellular lysates were analyzed for GSH levels with GSSG/GSH quantification kit. Data are mean ± S.E. (n = 3; **P < 0.01 vs. control). (B,C) Effect of ACR on the protein level of SLC7A11. TM4 cells were incubated with the indicated concentrations of ACR for 3 h, and the cellular lysates were used for protein expression of SLC7A11 (B). Densitometric quantitation of these bands was shown in (C). Data in (C) are mean ± S.E. (n = 3; **P < 0.01). (D,E) Effect of ACR on intracellular Fe²⁺ level. TM4 cells were stimulated with the indicated concentrations of ACR for 3 h. FerroOrange staining was conducted for Fe²⁺ detection. (F,G) Induction of lipid peroxidation by ACR. TM4 cells were cultured with 100 µM ACR for 3 h, followed by staining with C11 BODIPY 581/591 for detection of lipid oxidation. Densitometric quantitation of relative fluorescence intensity in (D,F) was performed on 20 cells randomly selected from each group, and the data were shown in (E,G), respectively. (n = 20; **P < 0.01). (H,I) Effect of DFO on ACR-induced Sertoli cell death. TM4 cells were pretreated with or without 400 µM DFO for 30 min before exposing to 100 µM ACR for an additional 6 h. The cell viability was determined by Calcein-AM/PI staining (H) and WST assay (I). Data in (I) are mean ± S.E. (n = 5; **P < 0.01).