<i>LPAR1</i> (R163W) mutation promotes cell mobility through activation of Rho pathway.

<p>(<b>A</b>) Rho activation assays (upper panel) showed a transient increase of GTP-bound Rho after exposure to LPA ligand (10 μM) in both wild-type (WT) and mutant (MT) LPAR1 expressing NIH3T3 cells indicating activation of Rho pathway mediated by the receptors. Signal of immunobands in the Western blot (upper panel) was quantified, and abundance of the GTP-bound Rho is plotted after normalization against total Rho and β-actin in the lower panel. (<b>B</b>) MT LPAR1 showed heightened signaling through the Rho pathway in transiently transfected COS-7 cells exposed to LPA in a dose-response way (<i>P</i><0.05) compared to WT. SRE represents the serum response element activity normalized against renilla luciferase and vehicle control. Error bars represent standard errors. </p

Whole genome sequencing of tumor Met2 and paired germline DNA revealed extensive somatic alterations in tumor DNA.

<p>(<b>A</b>) A CIRCOS plot shows non-synonymous somatic mutations, copy number changes, lesser allele fraction, loss of heterozygosity (LOH), and abnormal junctions in the Met2 tumor genome. (<b>B</b>,<b>C</b>) Chromothripsis was evident by massive complex rearrangements detected at chromosomes 4q and 13p by whole genome sequencing. In the junction plots, black, green, and red lines represent deletions, tandem duplication, and inversion respectively. Each dot in the copy number and LAF plots represents a 2 kilobases DNA fragment, and red and green lines mark the average segmental copy number and LAF respectively. LAF, lesser allele fraction. </p

A focal hemizygous deletion in the <i>ATRX</i> gene in all three tumor samples.

<p>(<b>A</b>) Base coverage of X chromosome from whole genome sequencing detected a hemizygous deletion in the <i>ATRX</i> genes from 76916706 to 76932433 (marked by red arrows). Each black dot represents a base, and red lines denote average coverage of each segment using circular binary segmentation[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077731#B67" target="_blank">67</a>]. Transcript NM_000489 was used for the exon annotation of the <i>ATRX</i> gene. (<b>B</b>) Genomic PCR verified the somatic deletion in the <i>ATRX</i> gene using a pair of genomic PCR primers flanking the predicted deleted region on the X chromosome. As result of the deletion, an abnormal PCR product of 220bp was detected in tumor DNAs, but not in the germline DNAs on a 2% TBE-agarose gel. (<b>C</b>) Sanger sequencing of the genomic PCR products confirmed the deletion of ChrX:76916706-76932433 was the same among all three tumors .</p