Gestational and Peri-conceptional Exposure to Intra-nasal Instilled Air Pollutants Epigenetically Perturb Metabolic, Placental and Embryonic Phenotypes

<p><strong>Supplementary Figure legends</strong></p> <p>&nbsp;</p> <p><strong>Figure 1: </strong>Gestational study: Bar plots of plasma metabolites</p> <p>&nbsp;</p> <p><strong>Figure 2:</strong> Gestational study: Bar graph depicting gestational day 19 (GD19) maternal (left) and fetal (right) blood glucose concentrations presented as mean &plusmn; SEM. Air pollutant exposed group (AP) versus controls (CON), *p &lt; 0.05 AP versus CON.</p> <p>&nbsp;</p> <p><strong>Figure 3: </strong>Gestational study: <strong>(A)</strong> GO term enrichment analysis of expressed genes between the AP group versus the CON group (n=6 each). Significant enrichment of pathways represented as q-values have been presented in a color-coded key. NES = normalized enrichment score. <strong>(B)</strong> Category cnet plot depicting the linkage of genes and biological concepts (hallmark pathways) as a network illustrating which genes are involved in enriched pathways, and genes that may belong to multiple annotation categories.</p> <p><strong>Figure 4: </strong>Gestational study:<strong> (A) </strong>Representative Western blots of various glucose and lipid transporter proteins and TNF&alpha; protein, with vinculin serving as an internal loading control, obtained from CON and AP GD19 placentas<strong>. (B) </strong>Bar plot showing densitometric analysis of Western blots of the various glucose and lipid transporters and TNF&alpha; (n=8 for each protein in each group). Data are depicted as Mean &plusmn; SEM. While trends are seen, statistical significance was not achieved.</p> <p><strong>Figure 5: </strong>Periconceptional study: <strong>(A)</strong> Bar plots demonstrating fasting blood glucose concentrations (left panel), Glucose tolerance tests (GTTs) (middle panel) and the area under the curves (AUCs) for GTTs (right panel) in non-pregnant (NP), pregnant (P), omega-3 diet exposed (P<em>n-3</em>) pregnant C57/BL6 (BL6) and CD1 pregnant mice are shown. <strong>(B)</strong> Bar plots demonstrating basal fed-state blood glucose concentrations (left panel), Insulin tolerance tests (ITTs) (middle panel) and AUCs for ITTs (right panel) in non-pregnant (NP), pregnant (P), omega-3 diet exposed (P<em>n-3</em>) pregnant C57/BL6 (BL6) and CD1 pregnant mice are shown (n=4-6 each). Data are presented as Mean &plusmn; SEM. Significance is shown as ^#compared to BL6-NP, *compared to BL6-P, and ^!compared to BL6-Pn-3 at a p&lt; 0.05.</p> <p><strong>Figure 6: </strong>Periconceptional study:<strong> (A) </strong>Representative Western blots demonstrate various GD19 placental glucose and lipid transporter proteins, with vinculin serving as the internal loading control. <strong>(B) </strong>Bar plots show densitometric analysis of the Western blots of the various glucose and lipid transporters (n=8 for each protein in each group). Data are presented as Mean &plusmn; SEM.</p> <p><strong>Figure 7</strong>: Periconceptional study: Category cnet plot depicting the linkage of genes and biological concepts (hallmark pathways) as a network illustrating which genes are involved in enriched pathways, and the genes that may belong to multiple annotation categories between <strong>(A)</strong> AP and CON,<strong> (B)</strong> AP-<em>n-3</em> and AP, <strong>(C) </strong>AP-<em>n-3</em> and CON.</p> <p><strong>Figure 8: </strong>Periconceptional study:<strong> (A)</strong> Bar plots showing coverage of the 5&rsquo;-hydroxymethylation DNA marks and genomic annotations in GD19 placentas from CON, AP, <em>n-3</em> and AP<em>-n-3</em> experimental groups (n=4 each). <strong>(B)</strong> Pie charts showing the distribution and genomic annotation of differentially methylated regions (DMRs) between the AP and CON groups. Upper panel depicts the distribution of DMRs across CpG-islands and associated regions, while the lower panel depicts the distribution of DMRs around different genomic regions. <strong>(C)</strong> Pie charts showing the distribution and genomic annotation of differentially methylated regions (DMRs) between AP<em>-n-3</em> and CON (left panel), <em>n-3</em> versus CON (middle panel) and AP<em>-n-3</em> versus AP (right panel).</p> <p><strong>Figure 9:</strong> Periconceptional study: <strong>(A-D) </strong>Volcano plots demonstrating the DNA methylation profiles of GD19 placentas from <strong>(A)</strong> AP versus CON, <strong>(B)</strong> AP<em>n-3</em> vs CON, <strong>(C)</strong> <em>n-3</em> vs CON, and <strong>(D)</strong> AP-<em>n-3</em> vs AP groups. Red and blue dots represent hypomethylated and hypermethylated loci respectively (n=4 each group).&nbsp;&nbsp;</p> <p>&nbsp;</p> <p>&nbsp;</p> <p>&nbsp;</p> <p>&nbsp;</p>