Workflow.

<p>The figure illustrates the procedure of the present study determining microRNA and TF co-regulation in OS cell proliferation.</p

Additional file 1: of The CXCR4 antagonist plerixafor (AMD3100) promotes proliferation of Ewing sarcoma cell lines in vitro and activates receptor tyrosine kinase signaling

Figure S1. Granulocyte-colony stimulating factor and DMSO vehicle do not induce proliferation of Ewing sarcoma cell lines in vitro. Figure S2. Serum-deprivation does not alter cell line groups of CXCR4-high and -low surface expressions. (PDF 625 kb

Summary of proliferation-related microRNA and TF co-regulations.

<p>The table specifies the number of common and interacting target genes between non-random microRNA and TF pairs for 3-node and 4-node motifs and for the total co-regulatory network they form.</p

MicroRNA and TF co-regulatory motifs.

<p>Schematic illustration of (<b>A</b>) 3-node and (<b>B</b>) 4-node FFL motifs. A 3-node motif contains a microRNA, a TF, and a commonly regulated target gene. In contrast, 4-node FFLs comprise a microRNA, a TF, a microRNA target gene (primary target), and a TF target gene (secondary target) that interacts with the primary target. MicroRNAs are indicated with diamonds, TF with triangles, primary targets with rectangles, and secondary targets with circles. (<b>C</b>) Coexpression of microRNA and TF target genes. The plot shows the observed and random cumulative distribution functions (CDFs, y-axis) of the Pearson correlation coefficients (x-axis) between any gene pair regulated by a specific microRNA and TF 3-node and 4-node motif. The observed and random CDFs are compared using the KS test. The p-values are indicated within the plots. Green and orange color codes correspond to the distinct clusters C1 and C2.</p

Differentially expressed microRNAs dependent on OS cell proliferation.

<p>DE microRNAs are shown with their corresponding log2 FC and FDR. A negative log2 FC indicates that microRNAs are down-regulated in high proliferative OS cells and vice versa. The references demonstrate an implication in cancer of the respective microRNA.</p

Proliferation and migration potential of OS cell lines.

<p>Proliferation and migration potential of OS cell lines.</p

Functional implications of microRNA target genes in OS proliferation.

<p>(<b>A</b>) Differentially expressed microRNA target genes. The heatmap shows the expression of DE microRNA target genes (log2 FC≥|0.7| & p-value<0.05, y-axis) among all OS cell samples (x-axis). High (dark blue) and low (light blue) proliferative OS samples are grouped into distinct clusters. The red/green color code corresponds to the expression deviation from the average expression among all samples. Complete-linkage clustering was applied with the Pearson correlation as distance metric. (<b>B</b>) Principal component analysis based on GO functional similarity. The figure draws the first (x-axis) and second (y-axis) principal components based on the GO functional similarity of microRNA target genes. The target genes are color coded according to their cluster membership determined by FCM. The radius of each gene represents the strength of association to its cluster. (<b>C</b>) Comparison of the GO enrichment analyses of the distinct clusters. Each row corresponds to a specific biological process category. The number of genes included in each category is indicated in the round brackets. Circle sizes correspond to the percentage of row-genes located in a cluster. Significantly enriched categories are color coded in red and random categories in blue.</p

Model of OS cell proliferation.

<p>The picture illustrates the proposed model of OS cell proliferation co-regulated by <i>miR-9-5p</i>, <i>miR-138</i>, and <i>miR-214</i> and the TFs <i>SP1</i> and <i>MYC</i>. These regulators control mainly components of the <i>NFKB</i>- and <i>RB1</i>-pathway and of focal adhesion processes. Primary microRNA targets are rectangular and secondary targets are ellipse shaped. Red marks up-regulated genes in proliferative active OS cell lines and green indicates down-regulated genes. White nodes represent genes not DE in OS cell proliferation. Solid and dashed arrows illustrate direct and indirect functional associations, respectively. <i>RB1</i> is shaded in red to indicate that it is no member of the global networks.</p

<i>miR-9-5p</i> and <i>SP1</i> co-regulatory motifs.

<p>The co-regulatory motifs of <i>miR-9-5p</i> and <i>SP1</i> are illustrated as graphs with nodes and edges for (<b>A</b>) C1 and (<b>B</b>) C2. MicroRNAs are marked with diamonds, TFs with triangles, primary targets with rectangle, and secondary targets with a circle. Yellow edges tag protein-DNA interactions, blue edges microRNA-target interactions, and dashed edges protein interactions. The red/green color code presents the corresponding log2 FC.</p

Additional file 2: of The CXCR4 antagonist plerixafor (AMD3100) promotes proliferation of Ewing sarcoma cell lines in vitro and activates receptor tyrosine kinase signaling

Table S1. Plerixafor treatment changes receptor tyrosine kinase phosphorylation patterns in CXCR4-high and -low Ewing sarcoma cell lines. (PDF 1503 kb