Stage-Specific Gene Co-Expression Networks via WGCNA. (A) PCA analysis of all RNA sequencing samples. (B) Pearson correlation co-efficient analysis of all RNA sequencing samples. (C) Hierarchical clustering information of all RNA sequencing samples. (D) The determination of soft threshold power, when β = 6, the scale-free network fitting. (E) Based on the hierarchical clustering and adjacency dissimilarity, a gene clustering tree diagram of 22 modules was obtained. (F) Module-sample relationship, where the horizontal axis represents the samples and the vertical axis represents the modules. The numbers in each grid represent the correlation between the modules and the samples.

Networks of genes in different modules. (A) The key hub genes were shown in Yellow Module (ncapd2, smarcc1a, kif15). (B) The key hub genes were shown in Magenta Module (hunk, pbx1a, zc2hc1a, lypd6, sich211-235e15.1). (C) The key hub genes were shown in Lightgreen Module (irg1l, sich211-153b23.5, noxo1a, sich211-153b23.3, zgc:100868). (D) The key hub genes were shown in Magenta Module (chrna6, slc4a10a, slc6a11b). (E,F) Gene expression of the key hub genes in 4 modules by RNA-seq (E) and qRT-PCR (F). Value are normalized to Control group, and represent mean ± SE from three independent experiments, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001 (Student’s t-test).

The neurotoxicity and Ferroptosis identified in zebrafish embryos. (A) Neuron system fluorescence signals of control group and ESIONPs-exposed group in transgenic zebrafish Tg (eef1a1l1:EGFP). (B) Polarized light intensity of control group and ESIONPs-exposed group of zebrafish muscles. (C) The expression of neuron-developmental markers (pax2a, neurog1, and axin2) at different developmental stages of zebrafish. (D) The expression of muscle markers (acta2, ttn, lpin1) at different developmental stages of zebrafish. (E) ROS generation in zebrafish embryos was detected with fluorescent probe DCFH-DA staining. (F) The prevalence of apoptosis in zebrafish embryos was detected with fluorescent dye AO staining; red arrows indicate the apoptotic cells. (G) The expression of oxidative stress biomarkers (cybb, nox1, rac2) at different developmental stages of zebrafish. (H) The expression of apoptosis biomarkers (jnk1, bcl2a, tp53) at different developmental stages of zebrafish. The fluorescence intensity and polarized light intensity was quantified for individual zebrafish using ImageJ analysis. Scale bar, 200 μm. Value are normalized to Control group, and represent mean ± SE from three independent experiments, * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001 (Student’s t-test).