Fig. 6

Therapeutic effects of 3-HPAA on spermatogenesis are modulated by GPX-4-mediated ferroptosis. AB Typical scatter plots (A) and summary (B) of Annexin V-FITC/PI assay of GC-2 cells in vehicle, H₂O₂, and 3-HPAA groups. GC-2 cells were cultured in normal medium (labeled as vehicle) or treated with 200 μM hydrogen peroxide (H₂O₂) for 90 min, and then 50 μM of 3-HPAA (labeled as 3-HPAA) or 0.1% DMSO (labeled as H₂O₂) was added into the medium. 48 h later, cells were harvested for subsequent experiments. C ROS level of GC-2 cells in three groups. Left: representative image; right: statistics of the ROS level. D–F Oxidative stress-related indicators including TEAC (trolox-equivalent antioxidant capacity) (D), GPx (E), and MDA (F) in the three groups. G, H Protein expression of FTL, FTH1, GPX4, ACSL4, NRF2, and xCT in the three groups. I Representative images of the iron staining of GC-2 cells after different treatment. Fe²⁺ was detected using FerroOrange assay. J, K Western blot analysis of GPX4 (J) and iron staining (K) of GC-2 cells treated with RSL3 (200 nM). L, M Western blot analysis of GPX4 (L) and iron staining of GC-2 cells (M) treated with Gpx4 siRNA. Scale bars = 50 μm. White arrowheads indicated the Fe²⁺ signal-positive cell in I, K, and M. All data are presented as mean ± SEM. For B–H, P values were determined by one-way ANOVA with Sidak’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 compared to the vehicle or H₂O₂ group. For J and L, P values were determined by two-tailed unpaired Student’s t test. *P < 0.05. n = 3–6 per group