Fig. 5: H3K18la maintains granulosa cell proliferation and mitochondrial homeostasis.

A Cell viability of granulosa cells treated with control (CK), DON, DON + Nala (10 mM), or DON + lactate (10 mM) for 24 h, as determined by CCK8 assay (n = 6). B Immunoblot analysis of H3K18la levels in CK, DON-treated, DON + Nala (10 mM), and DON + lactate(10 mM)-treated groups, with H3 as the loading control. C qPCR analysis of GPX4 and ACSL4 gene expression levels in CK, DON-treated, and DON + Nala-treated granulosa cells (n = 3). D Representative confocal microscopy images of Fe²⁺ and ROS levels in CK, DON-treated, and DON + Nala-treated granulosa cells. (Scale bar: 40 µm). E Confocal microscopy images showing the intensity of the lipid peroxidation probe (BDP 581/591 C11) in granulosa cells of different groups. (Scale bar:40 µm). F Quantitative data for relative fluorescence of Fe²⁺ and ROS staining. G Confocal microscopy images showing mitochondrial membrane potential (JC-1 probe) and nuclear co-localization in granulosa cells of different groups. (Scale bar: 20 µm). H Quantitative data for relative fluorescence of BDP 581/591 C11 and JC-1 staining. I TEM images showing mitochondrial morphology in granulosa cells of different groups (scale bar: 20 µm). J, K qPCR analysis of the effects of Nala supplementation on the expression levels of steroidogenesis and reproductive hormone-related genes (n = 3). L Western blot (WB) analysis of the effects of Nala supplementation on the protein expression levels of steroidogenesis and reproductive hormone-related genes. M WB analysis of the effects of DON + Nala co-treatment on GPX4 and ACSL4 expression. Significant changes in gene expression were observed in the treated groups compared to controls. Bars indicate the mean ± SD. Data were analyzed using one-way ANOVA (A, C, I, J) and two-way ANOVA (F, G) 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.