Figure 2.

NEDD4L overexpression promoted granulosa cell ferroptosis. (A and B) qRT-PCR and western blot analysis of NEDD4L mRNA and protein levels in GCs with and without NEDD4L overexpression (n = 3). ***P < 0.001. (C) The GC viability was assessed by the CCK-8 assay (n = 3). **P < 0.01. (D and E) The GC viability was assessed by FDA (n = 3). **P < 0.01. (E) The cell viability of NEDD4L-OE GCs was determined using the CCK-8 assay in the presence or absence of ZVAD-FMK (5 M), necrostatin-1 (10 M), or Fer-1 (2 M).0.05. **P < 0.01; ns, not significant. (F–I) The level of Fe²⁺, the content of MDA, the level of ROS, and the content of GSH in NEDD4L-OE GCs was analyzed by ELISA (n = 3) *P < 0.05, **P < 0.01.