Fig 2: The regulatory effects of miR-30b-5p and Pax3 on gene expression.(A) Artificial upregulation and downregulation of miR-30b-5p expression using mimics and inhibitors, respectively. Expression of miR-30b-5p, Pax3, and SLC7A11 in cells was assessed using PCR. NC: transfection of negative control of miRNA vectors; OV: miR-30b-5p overexpression; KO: miR-30b-5p knockdown. *p < 0.05, **p < 0.01, compared with the control group. (B and C) Luciferase reporter assay was performed to identify the putative binding sites (seed sequences) for hsa-miR-30b-5p in the 3'-UTR of pax3 and SLC7A11 mRNA, as predicted by the bioinformatic analysis webs, including TargetScan, miRanda, and PicTar. The wild-type (WT) pax3 and SLC7A11 3'-UTR, as well as their mutant versions (MT), with mutations in the putative binding sites, were synthesized and inserted into a pmirGLO vector at XhoI and NotI restriction sites. HTR-8/SVneo cells were transfected with the WT or MT constructs, along with either hsa-miR-10b-3p mimics or negative control. The activity of firefly luciferase was measured and normalized to that of Renilla luciferase. Pax3(W): wild-type pax3 constructs; Pax3(M1) and Pax3(M2): two mutant type of Pax3 constructs; SLC7A11(W) and SLC7A11 (M): wild-type and mutant type of Pax3 constructs, respectively; Mimics: hsa-miR-10b-3p mimics. *p < 0.05, **p < 0.01, compared with the Pax3(W) and SLC7A11(W) groups; #p < 0.05, ##p < 0.01, compared with the Pax3(W) + Mimics and SLC7A11(W) + Mimics groups. (D) ChIP assay was performed to determine whether Pax3 could bind to the promoters of SLC7A11, FPN1 and TFR1 genes. **p < 0.01, ***p < 0.001, compared with the IgG group. (E) Pax3 in HTR-8/SVneo and TEV-1 cells was knocked down, followed by the evaluation of protein levels of SLC7A11, FPN1, and TFR1 using western blot assay. Pax3 KO: Pax3 knockdown. *p < 0.05, **p < 0.01, compared with the control group.

Fig 3: Downregulated Pax3 probably resulted in reduced expression of FPN1 in PE placental tissues.Both GSE50783 and GSE44667 data sets provided the data of genes that were aberrantly expressed in placental tissues from PE patients and physiologically normal pregnancies. Transcript factors that were implicated in the transcription of FPN1 were predicted using a Bioinformatics analysis website (http://bioinfo.life.hust.edu.cn/hTFtarget#!/). We analyzed the intersection between the aberrantly expressed genes and transcript factors targeting FPN1. As indicated by the Venn diagram, three of the transcript factors (Pax3, POU3F2 and BHLHE40) were found to be aberrantly expressed in placental tissues from PE patients (A). Bioinformatics analysis website Jasper (http://jaspar2016.genereg.net/) gave the highest score on POU3F2 followed by Pax3 (B). We performed western blot to test the protein levels of these three transcript factors in placental tissues from PE patients and physiologically normal pregnancies (C). Pax3 protein level was down-regulated in PE placental tissues, while BHLHE40 protein level was significantly up-regulated. Results from immunohistochemistry assay (D) and GSE10588 data set (E) also confirmed the down-regulation of PAX3 in PE placental tissues. Another transcript factor-predicting websit, Jasper (http://jaspar2016.genereg.net/), showed the motif, a sequence in gene promoter that is recognized by Pax3 (F). Moreover, Jasper revealed two potential binding sites of transcription factor Pax3 at the promoter of FPN1 and SLC7A11 genes. Our following study was performed to identify the regulatory effect of Pax3 on FPN1 and SLC7A11 expression. *p < 0.05, **p < 0.01, compared with the normal group.

Fig 4: Eriodictyol inhibits ferroptosis induced by the Aß1–42 oligomer in HT-22 cells. A The levels of ferrous iron and total iron in HT-22 cells were detected using an iron assay kit. B The iron content in HT-22 cells was determined using an immunofluorometric assay with calcein-AM.C The expression of TfRC, FTH, and Fpn was measured using Western blotting. D The relative mRNA expression levels of these proteins were tested using q-PCR. E ROS levels in HT-22 cells were measured using two types of fluorescent probes (DHE and DCFH-DA) and F the MDA content in HT-22 cells was measured using a Lipid Peroxidation MDA Assay Kit. G GPX4 protein levels in HT-22 cells were detected using Western blotting and H its relative mRNA expression level was measured using q-PCR. I The morphological features of mitochondria in HT-22 cells were observed under an electron microscope. The data are presented as the means ± SD of three experiments. **P < 0.01, and ***P < 0.001 compared with the Aß1–42 oligomer group. ##P < 0.01, ###P < 0.001 compared with the control group

Fig 5: Effects of organic trace elements on messenger RNA (mRNA) expression of duodenal trace element transporter in late-phase laying hens. DMT1 (A), divalent metal transporter 1; ZnT-1 (B), zinc transporter 1; and FPN1 (C), ferroportin 1. Different letters represent significant differences (p < 0.05).