<i>Tgfbr3</i>^<i>-/-</i> epicardial cells fail to activate the NF-ĸB signaling pathway.

<p>(A) (TOP) Genes dysregulated in each group (>2-fold, p<0.001) were counted. (BOTTOM) Shared targets of NF-ĸB signaling dyregulated in all groups are shown. Red<b>—</b>expressed higher in<i>Tgfbr3</i>^<i>+/+</i>, Green<b>—</b>expressed higher in <i>Tgfbr3</i>^<i>-/-</i>. (B) Cells transfected with an NF-ĸB responsive SEAP reporter construct and incubated with VEH, TGFβ1, TGFβ2, or BMP2 revealed the inability of <i>Tgfbr3</i>^<i>-/-</i> cells to induce NF-ĸB signaling. (C) Incubation of <i>Tgfbr3</i>^<i>+/+</i> epicardial cells in a transwell invasion assay with an NF-ĸB inhibitor (BMS345541) significantly reduced invasion (* = p < .01) in response to ligands known to promote <i>Tgfbr3</i>-dependent invasion.</p

GO Analysis of Genes >2-fold Differentially Expressed Between Genotypes Unique to Specific Ligand Incubation Groups.

<p>GO Analysis of Genes >2-fold Differentially Expressed Between Genotypes Unique to Specific Ligand Incubation Groups.</p

Gene regulatory network analysis identifies NF-kB signaling as a central node.

<p>Genes >2-fold (p<0.001) differentially expressed between <i>Tgfbr3</i>^<i>+/+</i> and <i>Tgfbr3</i>^<i>-/-</i> epicardial cells incubated with either TGFβ2 (A) or BMP2 (B) were subjected to gene ontology analysis (using DAVID software, p<0.0001). (C-D) NF-ĸB signaling (orange circle) is a central node in representative networks generated by gene regulatory network analysis (using Ingenuity Pathway Analysis software). Green- expressed higher in<i>Tgfbr3</i>^<i>+/+</i>, Red- expressed higher in <i>Tgfbr3</i>^<i>-/-</i>. (E) The distribution of the predicted protein location in the cell is depicted (proteins with unknown location are not shown).</p

RNA-seq analysis identifies genes dysregulated in <i>Tgfbr3</i>^<i>-/-</i> epicardial cells.

<p>(A) (Left) The number of genes >2-fold (p<0.001) differentially expressed between <i>Tgfbr3</i>^<i>+/+</i> and <i>Tgfbr3</i>^<i>-/-</i> epicardial cells for each group. (Right) The number genes similarly dysregulated within selected groups that were also annotated in the IPA database are shown with genes found in each. (B) The number of overlapping genes >2-fold differentially regulated (p<0.001) was determined and mapped. 129 genes were similarly dysregulated across all groups. (C) (Top) Gene ontology analysis of these 129 genes by DAVID revealed a significant (p<0.0001) enrichment of genes associated with specific biological processes. emb.- embryonic. (Bottom) A representative network generated by gene regulatory network analysis of the 129 genes using Ingenuity Pathway Analysis software is depicted. Green- expressed higher in<i>Tgfbr3</i>^<i>+/+</i>, Red- expressed higher in <i>Tgfbr3</i>^<i>-/-</i>.</p

Immortalized epicardial cells undergo loss of epithelial character.

<p>(A) The epicardium undergoes EMT at E11.5–13.5. Subsequently, transforming epicardial cells invade the subepicardial space and myocardium towards forming coronary vessels. Blue- epicardium. Purple- endothelium. Yellow- smooth muscle cells. Red- myocardium. (B) Immortalized epicardial cells were derived from E11.5 <i>Tgfbr3</i>^<i>+/+</i> and <i>Tgfbr3</i>^-/- embryos which expressed a temperature-sensitive large T-antigen. (C) Immunohistochemistry of <i>Tgfbr3</i>^<i>+/+</i> or <i>Tgfbr3</i>^-/- immortalized epicardial cells after 72 hours incubation with TGFβ2 or vehicle. TGFβ2 increased expression of SM22α and form stress fibers in the enlarged, elongated cells. ZO1 becomes redistributed to the cytoplasm.</p

<i>Tgfbr3</i>^<i>-/-</i> epicardial cells have dysregulated proliferation, apoptosis, and invasion.

<p>(A) Summary of the phenotypes of <i>Tgfbr3</i>^<i>+/+</i> and <i>Tgfbr3</i>^<i>-/-</i> epicardial cells <i>in vitro</i>. EC—epithelial character, SM Diff.- smooth muscle differentiation. (B) RNA-seq analysis of <i>Tgfbr3</i>^<i>+/+</i> and <i>Tgfbr3</i>^<i>-/-</i> epicardial cells incubated with ligand for 72 hours. Reads—the total number of mapped sequences for each of the 8 groups (in duplicate). Genes—the total number of genes with a significant number of reads (>10) mapped.</p

<i>Tgfbr3</i>^<i>+/+</i> and <i>Tgfbr3</i>^<i>-/-</i> epicardial RNA-seq datasets confirm cell identity and differential ligand response.

<p>(A) Cells express epicardial markers. Mean normalized reads between replicates and standard error are depicted. (B) Smooth muscle markers are markedly induced with TGFβ1 and TGFβ2 compared to BMP2 incubation. Fold is relative to VEH for each genotype. (C) Endothelial or myocardial markers are not expressed at significant levels (< 2 normalized reads). Mean normalized reads between replicates and standard error are depicted. (D) Genes >2-fold differentially expressed after ligand treatment compared to vehicle are depicted. Fewer genes are induced by incubation with TGFβ1–2 treatment in <i>Tgfbr3</i>^<i>-/-</i> epicardial cells compared to <i>Tgfbr3</i>^<i>+/+</i>, while the opposite is true with BMP incubation.</p

Genetic distance between the JC virus sequences of each brain sample and MAD-1 sequence.

<p>The figure illustrates the genetic divergence between the JC virus sequences found in each PML brain and MAD-1 (red bar), the JC virus prototype sequence associated with PML. Each chord corresponds to a single nucleotide polymorphism. The presence of multiple JCV variants within each PML brain accounts for a remarkable degree of genetic variation and suggests that the genetic complexity of JC virus in the brain compartment has previously been underestimated.</p

ViroFind performance characteristics.

<p>ViroFind performance characteristics.</p

Multiple JC virus variants within the same PML brain sample.

<p>Visual inspection of the JC virus 75 base-pair nucleotide reads reveals multiple single nucleotide polymorphisms (SNP). Each read is represented by a single horizontal arrow. SNPs in positions 3,122, 3,178 and 3,185 of the JC virus genome occur independently, which is consistent with the presence of at least three distinct viral variants in the same PML brain.</p