Fig. 2: DON induces oxidative damage in porcine granulosa cells.

A Cell viability of porcine granulosa cells treated with different concentrations of DON (0–1 μg/ml) for 24 h, as determined by CCK8 assay (n = 6). B Morphological observation of porcine granulosa cells in the control and DON-treated groups (magnification 100×). C Representative images of ROS levels in granulosa cells from the control and DON-treated groups, observed by confocal microscopy. Scale bars: 40 µm. D–F ELISA measurement of ROS, GSH, and MDA levels (n = 6). G, H mRNA expression levels of steroidogenesis and reproductive hormone-related genes in porcine granulosa cells determined by qPCR (n = 3). I Protein expression levels of steroidogenesis and reproductive hormone-related genes in porcine granulosa cells determined by Western blot (WB), with Tubulin as the loading control (n = 3). J ELISA measurement of reproductive hormone E2 levels in porcine granulosa cells (n = 6). K Number of peptides detected in the two groups. L Score plot of principal component analysis (PCA) of proteomics data from control and DON-treated granulosa cells. M Volcano plot showing differentially expressed proteins. Red represents upregulated proteins, blue represents downregulated proteins, and gray represents no significant difference. N Heatmap showing the expression levels of antioxidant-related proteins in control and DON-treated granulosa cells (n = 3). O, P qPCR quantification of antioxidant genes SOD2 and CAT (n = 3). Bars indicate the mean ± SD. Statistical significance was analyzed using one-way ANOVA (A)and unpaired Student’s t-test (D–H, J, O, P). (ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).