Enzyme kinetics of both MTAP variants in stably transfected cells.

<p>Quantification of MTAP expression at the mRNA (A) and protein level (B) in the melanoma cell line Mel Juso after stable transfection of control plasmid (pCMX-PL1) or expression constructs for MTAP-56I and MTAP-56V. The original Western blot is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160348#pone.0160348.s003" target="_blank">S3 Fig</a>. (C) Analysis of intracellular MTA levels in the stably transfected Mel Juso cell clones.</p

Characterization of the Methylthioadenosine Phosphorylase Polymorphism rs7023954 - Incidence and Effects on Enzymatic Function in Malignant Melanoma

<div><p>Deficiency of methylthioadenosine phosphorylase (<i>MTAP</i>) supports melanoma development and progression through accumulation of its substrate 5’-methylthioadenosine (MTA), which leads amongst others to a constitutive inhibition of protein arginine methyltransferases (PRMTs) and activation of the transcription factor AP-1 via the receptor ADORA2B. Genetic association studies have also suggested that genetic polymorphism in <i>MTAP</i> may modulate the risk of melanoma. Here, we investigated the only globally common non-synonymous single nucleotide polymorphism (SNP) reported to date for <i>MTAP</i>. The SNP rs7023954 is located in exon 3 (c.166G>A), and leads to the conservative substitution of one branched-chain amino acid residue (valine) for another (isoleucine) at position 56 (p.Val56Ile). Whereas genotype frequencies in normal and primary melanoma tissues or cell lines were in Hardy-Weinberg equilibrium based on cDNA amplicon sequencing, a marked (P = 0.00019) deviation was observed in metastatic melanoma tissues and cell lines due to a deficit of heterozygotes. Enzyme assays conducted on the co-dominantly expressed alleles revealed no difference in the conversion rate of MTA to adenine and 5-methylthioribose-1-phosphate, indicating that this known enzymatic activity does not modulate the tumor suppressive function of MTAP.</p></div

Enzyme kinetics of both <i>in vitro</i>-translated MTAP variants.

<p>(A) Western Blot for determination of MTAP expression by <i>in vitro</i> transcription/translation of the pCMX-PL1 (1.0) control plasmid and the expression constructs for MTAP-56I (2.56-fold increased) and MTAP-56V (2.28-fold increased). Densitometric quantification revealed increased MTAP amount. The original Western blot is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160348#pone.0160348.s001" target="_blank">S1 Fig</a>. (B) Analysis of the metabolic activity of the <i>in vitro</i>-translated products by liquid chromatography–tandem mass spectrometry. Catalytic rates of IVT-MTAP-56I and IVT-MTAP-56V were corrected for the rate of the control, which was set at 1.</p

Determination of <i>MTAP</i> rs7023954 in cell lines and tissue samples.

<p>The non-synonymous <i>MTAP</i> SNP c.G166A was genotyped by sequencing of cDNA that had been produced from total RNA extracted from both normal as well as primary (prim.) and metastatic (met.) melanoma cell lines and tissues. Distribution of the genotypes AA, AG, and GG is shown. A detailed description of the genotypes of the samples investigated is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160348#pone.0160348.s004" target="_blank">S1 Table</a>.</p

Continuous Water Infusion Enhances Atmospheric Pressure Chemical Ionization of Methyl Chloroformate Derivatives in Gas Chromatography Coupled to Time-of-Flight Mass Spectrometry-Based Metabolomics

The effects of continuous water infusion on efficiency and repeatability of atmospheric pressure chemical ionization of both methyl chloroformate (MCF) and methoxime-trimethylsilyl (MO-TMS) derivatives of metabolites were evaluated using gas chromatography–time-of-flight mass spectrometry. Water infusion at a flow-rate of 0.4 mL/h yielded not only an average 16.6-fold increase in intensity of the quasimolecular ion for 20 MCF-derivatized metabolite standards through suppression of in-source fragmentation but also the most repeatable peak area integrals. The impact of water infusion was the greatest for dicarboxylic acids and the least for (hetero-) aromatic compounds. Water infusion also improved the ability to detect reliably fold changes as small as 1.33-fold for the same 20 MCF-derivatized metabolite standards spiked into a human serum extract. On the other hand, MO-TMS derivatives were not significantly affected by water infusion, neither in their fragmentation patterns nor with regard to the detection of differentially regulated compounds. As a proof of principle, we applied MCF derivatization and GC-APCI-TOFMS to the detection of changes in abundance of metabolites in pancreatic cancer cells upon treatment with 17-DMAG. Water infusion increased not only the number of metabolites identified via their quasimolecular ion but also the reproducibility of peak areas, thereby almost doubling the number of significantly regulated metabolites (false discovery rate < 0.05) to a total of 23

Effect of both MTAP variants in transiently cells.

<p>Quantification of MTAP expression at the mRNA (A) and protein level (B) in the melanoma cell line Mel Juso after transient transfection of expression constructs for MTAP-56I and MTAP-56V or control plasmid (pCMX-PL1). The original Western blot is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160348#pone.0160348.s002" target="_blank">S2 Fig</a>. (C) Analysis of intracellular MTA levels in the transiently transfected Mel Juso cells.</p

Confirmation of cDNA genotype by liquid chromatography-tandem mass spectrometry (LC-MS/MS) based protein expression analysis.

<p>Sole expression of the 56I-allele was observed in melanoma cell lines Mel Im, Mel Ho, and A375, whereas the 56V-allele was detected exclusively in WM793, WM1366, and WM293A. In the fibroblast cell line 3F0379, both alleles were co-expressed equally.</p

COLXVI overexpression induces MMP9 expression.

<p>(<b>A</b>) Immunoblot analysis of collagen XVI secretion in supernatants of COLXVI cell clones (clones 1-4) and mock control cells (mock 1-2). Only COLXVI cell clones secret the full-length form of COLXVI (213 kDa; black arrow). Clones 3 and 4 exhibit higher COLXVI secretion than clones 1 and 2. COLXVI cell clones also secrete collagen XVI fragments. A Coomassie Blue membrane staining was used as loading control. (<b>B</b>) Quantitative PCR of <i>MMP9</i> expression in COLXVI cell clones and mocks after 24 h incubation with/without recombinant collagen XVI. COLXVI cell clones (1-4) show a significant expression of <i>MMP9</i> that is further enhanced by the addition of recombinant collagen XVI (n = 3). (<b>C</b>) Gelatin zymography of COLXVI cell supernatant and mock controls. The COLXVI cell clones (1-4) show a clear gelatinolytic activity at 92 kDa (pro form). In contrast, the mock control cells (1-2) show very weak MMP9 bands. (<b>D</b>) Immunoblot of total ILK and phosphorylated ILK (P ILK) isolated from membrane fractions of COLXVI cell clones and mock control cells. ILK is activated in COLXVI cell clones whereas in mock control cells P-ILK is lacking. (<b>E</b>) Quantitative PCR of <i>MMP9</i> expression after ILK inhibition with Cpd 22 (ILKi). The expression of <i>MMP9</i> decreased after ILK inhibition in the COLXVI cell clones. (n = 3). (<b>F</b>) Gelatin zymography of the supernatant of the COLXVI cell clone 3 after ILK inhibition with Cpd 22 (ILKi; c  =  300nM). After ILK inhibition the COLXVI cell clone depicts decreased MMP9 secretion. (<b>G</b>) Promoter activity of the COLXVI cell clone 3 after ILK inhibition with Cpd 22 (ILKi; c  =  300 nM). ILK inhibition results in a significant decrease of the MMP9b promoter activity. (p<0.001; n = 3). (<b>H</b>) Immunoblot of total Akt/PKB and phosphorylated Akt/PKB (P-Akt/PKB) in cell lysates of COLXVI cell clones (clones 1-4) and mock control cells (1-2). Akt/PKB activation is increased in COLXVI cell clones compared to mock controls.</p

Collagen XVI overexpression leads to increased invasion of OSCC cells.

<p>(<b>A</b>) Quantitative PCR of <i>MMP9</i> gene expression of COLXVI and mock control cells after AP-1 inhibition with Tanshinone IIA (TIIA; c  =  100 ng/mL). The expression of <i>MMP9</i> decreased in the COLXVI cell clones after AP-1 inhibition with Tanshinone IIA. (n = 3) (<b>B</b>) 3D micromass pellets of COLXVI cells compared to mock control cells 24 h after placement. COLXVI cells (high and low expressing) show a significantly wider invasion zone (orange line) compared to mock control cells (p<0.01; n = 10). (<b>C</b>) 3D micromass pellets of mock control cells after 24 h of incubation with 500 ng/mL recombinant collagen XVI. Incubation with recombinant collagen XVI resulted in a significantly increased spreading of the invasion zone (orange line) compared to the control (p<0.001; n = 10).</p

Collagen XVI overexpression leads to an increase of ILK/kindlin-1 interaction.

<p>(<b>A</b>) Proximity ligation assay for the analysis of ILK/kindlin-1 interaction in COLXVI and mock control cells (scale bar equals 50 µm). COLXVI cell clones exhibit an increased interaction of kindlin-1 and ILK at focal adhesions compared to mock control cells (<b>A+B</b>). After inhibition of focal adhesion formation with soluble RGD peptides (c  =  100 µg/mL) ILK/kindlin-1 interaction was significantly reduced (p<0.001, n = 100) (A+<b>C</b>). (<b>D</b>) Quantitative PCR of <i>MMP9</i> gene expression in COLXVI cell clones and mocks after 24 h incubation with soluble RGD peptides. Inhibition of focal adhesions via soluble RGD-peptides resulted in a dose-dependent decrease in <i>MMP9</i> gene expression (n = 3). (<b>E</b>) Co-Immunoprecipitation of ILK and kindlin-1 of protein extracts isolated from COLXVI clones and mocks. COLXVI cell clones exhibit an increased ILK/kindlin-1 interaction depending on collagen XVI dose. Mock control cells showed least ILK/kindlin-1 interaction.</p