Knockdown of PLXNA1 impairs migration of human ERMS cells.

<p>Representative images of ERMS cells transfected with gene-specific siRNAs at 0 hr (A, control siRNA; C, <i>CCND2</i> siRNA; E, <i>HOXC6</i> siRNA; G, <i>PLXNA1</i> siRNA) and 22 hrs (B, control siRNA; D, <i>CCND2</i> siRNA; F, <i>HOXC6</i> siRNA; H, <i>PLXNA1</i> siRNA) following gap creation. Scale bar indicates 100 µm. (I) Quantification of data from wound healing assay. Each error bar indicates standard deviation across 5–6 independent replicates. (J) A Transwell migration assay was performed in RD cells that stably express either a control shRNA or two independent <i>PLXNA1</i> shRNAs. Migration was assessed after 24 hours. Each error bar indicates standard deviation across six fields at 200× magnification. Asterisks denote p<0. 05.</p

Knockdown of PLXNA1 induced differentiation and impaired anchorage-independent growth of human ERMS cells.

<p>RD cells stained with myosin heavy chain (MF20) and DAPI following culture under differentiation conditions for 72 hrs. (A) Control siRNA. (B) <i>PLXNA1</i> smart-pool siRNA. (C) Control scrambled shRNA. (D) <i>PLXNA1</i> shRNA-1. DAPI, blue; MF20-positive cell, green. (E) Quantification of MF-20 immunofluorescence in siRNA and shRNA-knockdown RD cells. Asterisk indicates significant differences between gene knock- down and control cells (p<0.05). Error bars denote standard deviation. (F) Western analysis of <i>PLXNA1</i> shRNA stable knockdown; sc, scrambled control shRNA; 1, <i>PLXNA1</i> shRNA-1; 2, <i>PLXNA1</i> shRNA-2. A soft agar colony formation assay to assess PLXNA1 knockdown effects on anchorage-independent growth (G–I). (G) Control scrambled shRNA. (H) <i>PLXNA1</i> shRNA. (I) Quantification of colony formation assay results. Error bar indicates standard deviation from triplicate experiments.</p

Chemical inhibition of VEGF signaling by cediranib reduces ERMS growth <i>in vivo</i>.

<p>Syngeneic CG1 fish were transplanted with ERMS cells that co-expressed <i>rag2-KRASG12D</i> and <i>rag2-dsRED</i>. Fish with engrafted tumors were treated with DMSO vehicle (A–F) or 100 nM of cediranib for 7 days (G–L). Pre-treatment (A–C and G–I) and post-treatment images (D–F and J–L) of representative fish. Bright field (A,D,G,J), dsRED fluorescence (B,E,H,K) and merged image planes (C,F,I,L). Scale bar is 3 mm. (M) Quantification of relative volume change for individual animals. (N–O) <i>fli1-GFP</i> transgenic zebrafish were transplanted with dsRED-labeled ERMS and treated with DMSO (N) and cediranib (O). Scale bar equals 50 µm. (P) Microvessel density quantification. Asterisk indicates statistically significant difference between DMSO and cediranib-treated groups based on student t-test. Each error bar indicates standard deviation from 3 fields of microvessels for each animal. EDU incorporation analysis in DMSO (Q) or cediranib (R) treated fish. Scale bar is 50 µm. (S) Quantification of EDU analysis across each cohort of animals. Each error bar indicates standard deviation of percent EDU+ cells found within 3 fields for each animal.</p

Array CGH reveals cancer-specific chromosomal abnormalities in zebrafish ERMS.

<p>(A) Summary of common gene-containing CNA gains (green) and losses (red) in 20 animals examined. Only recurrent CNAs found in ≥3 samples are shown. The height of each bar correlates with the frequency of each aberration. Detailed view of regional gains for <i>vegfa</i> on chromosome 4 (B), <i>ccnd2a</i> on chromosome 25 (C), <i>hoxc6a</i> on chromosome 23 (D), and <i>plxna1</i> on chromosome 6 (E). Y-axis denotes log2 ratio of the probes and X-axis denotes genomic coordinates.</p

Comparison of array CGH analyses in zebrafish and human ERMS.

<p>Zebrafish gene-containing, CNAs were compared to those identified in 26 primary human ERMS samples by Paulson et al. (2011). Genes selected for characterization in this study are in bold.</p