Fig. 5: Identification and verification of reliable biomarkers to predict male reproductive toxicity in human Sertoli cells.

The major steps for the RNA-sequencing protocol, including the experimental design, read alignment, quantification, and visualization, are presented as a schematic (a). The large-scale RNA sequencing data are shown as a heatmap of differential gene expression in the control groups compared to those treated with low (5 ng/ml) and high (7 ng/ml) concentrations of dioxin; increased (red) or decreased (green) gene expression compared to the mRNA levels in the control groups is indicated (b). KEGG pathway analysis was subsequently conducted to determine which pathways and functions are likely to be associated with toxin exposure (c). Among the differentially expressed genes, a positive correlation was found between the significantly increased SERPINB2 expression levels and toxin exposure in human Sertoli cells (d, e). Real-time PCR and western blotting were used to verify that the levels of SERPINB2 increased after treatment with low and high concentrations of toxin (f). β-actin was used as the internal protein control, and PPIA was used as the housekeeping gene for real-time PCR. All experiments were performed in triplicate. The data are presented as the means ± SDs. *p < 0.05; **p < 0.005; and ***p < 0.001 (two-sample t test).