Fig. 3: DON induces ferroptosis in porcine ovarian granulosa cells.

A GO cellular component enrichment analysis of differentially expressed proteins in control (CK) and DON-treated granulosa cells. The size of the dots represents the number of proteins, and the color represents the significance of the pathway. The x-axis indicates the enrichment factor, and the y-axis represents different functional terms. B–D Western blot (WB) and immunofluorescence analysis of GPX4 and ACSL4 protein abundance in CK and DON-treated granulosa cells. Scale bars: 40 µm. E Transmission electron microscopy (TEM) images showing mitochondrial morphology in granulosa cells before and after DON treatment (scale bar: 4 µm and 0.4 µm). F, G Representative images of Fe²⁺ and ROS levels and their relative fluorescence intensity in CK, DON-treated, and Fer-1-treated granulosa cells observed by laser confocal microscopy (n = 3). Scale bars: 40 µm. H, I Representative images of mitochondrial membrane potential (JC-1 probe) levels and their relative fluorescence intensity in CK, DON-treated, and Fer-1-treated granulosa cells observed by laser confocal microscopy (n = 3). Scale bars: 40 µm. Red represents high mitochondrial membrane potential, green represents low mitochondrial membrane potential, and blue represents nuclei. Scale bars: 40 µm. J–L WB analysis of GPX4 and ACSL4 protein expression levels in different groups(n = 3). M–S qPCR analysis of mRNA expression levels of reproduction-related genes of different groups (n = 3). Bars indicate the mean ± SD. Data were analyzed using two-way ANOVA (G) or one-way ANOVA (I, K–S) with Tukey’s post hoc test, with significance levels denoted as follows: ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.