FIGURE 4.
Inhibition of H2S promoted ferroptosis in ACR-treated Sertoli cells. (A,B) Acceleration of Sertoli cell death by BCA. TM4 cells were pretreated with or without 2 mM BCA for 12 h before exposing to 50 µM ACR for an additional 5 h. The cell viability was determined by Calcein-AM/PI staining (A) and WST assay (B). Data in B are mean ± S.E. (n = 5; **P < 0.01). (C) Aggravation of protein carbonylation and P38 activation by BCA. TM4 cells were exposed to 50 µM ACR with or without 2 mM BCA for 2 h. Cellular lysates were subjected to analysis of protein carbonylation and P38 phosphorylation. (D) Acceleration of GSH consumption by BCA. TM4 cells were pretreated with or without 2 mM BCA for 6 h before exposing to 50 µM ACR for an additional 3 h. The cellular lysates were analyzed for GSH levels with GSSG/GSH quantification kit. Data are mean ± S.E. (n = 4; **P < 0.01). (E,F) Aggravation of downregulated protein expression of SLC7A11 by BCA. TM4 cells were incubated with 75 µM ACR for 3 h, and the cellular lysates were used for protein expression of SLC7A11 (E). Densitometric quantitation of these bands was shown in (F). Data in (F) are mean ± S.E. (n = 3; *P < 0.05, **P < 0.01). (G,H) Promotion of lipid peroxidation by BCA. TM4 cells were exposed to 50 µM ACR with or without 2 mM BCA pretreatment for 3 h. After that, C11 BODIPY 581/591 staining were used for analyzing lipid oxidation. Densitometric quantitation of relative fluorescence intensity in (G) was calculated on 20 cells randomly selected from each group, and the data were shown in (H) (n = 20; *P < 0.05, **P < 0.01).