Effect of PCs inhibition on uPA, uPAR and PAI-1 expression.

<p>(<b>A–C</b>), Following total RNA extraction from indicated cells, real-time PCR analysis was performed using specific primers for indicated genes as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009992#s2" target="_blank">Materials and Methods</a>. Results are shown in the bar graph and are expressed as the mRNA transcript ratios. Data are shown as means ± S.E of three experiments performed in duplicate. Student's <i>t</i> test was used for statistical analysis. *, <i>P</i><0.05 and ***, <i>P</i><0.001. (<b>D</b>), Tumor cells were incubated at 37°C in serum-free media. After 24 hours, media were collected and analyzed for the presence of uPAR using ELISA kit. Results shown are representative of 3 experiments. Data are mean ± SEM (<i>n = </i>6 per group). Student's <i>t</i> test was used for statistical analysis ***, <i>P</i><0.001</p

Immunohistochemical staining of EMMPRIN, VEGFR-2 and HIF-2α in sections of human melanoma tissues.

<p><b>Left</b>, representative melanoma with lower expression of EMMPRIN. (<b>A</b>) EMMPRIN, (<b>B</b>) VEGFR-2 and (<b>C</b>) HIF-2α staining. <b>Right</b>, representative melanoma exhibiting strong expression of EMMPRIN. (<b>D</b>) EMMPRIN, (<b>E</b>) VEGFR-2, and (<b>F</b>) HIF-2α staining.</p

Schematic representation of the effects of PCs inhibition on migration and invasion of human melanoma cells with altered <i>CDKN2A</i>, <i>p53</i> and <i>Ras</i> genes.

<p>The <i>CDKN2A</i> locus codes for the tumor suppressors p16 and ARF that regulate cell cycle progression, DNA repair and cell invasion via activating pRb, and the p53 pathway via inhibiting the activity of MDM2, respectively. Mutations or deletions of the <i>CDKN2A</i> gene result in altered MMPs/TIMPs and/or uPA/uPAR/PAI-1 expression and activity, which in turn lead to tumor cell invasion. The <i>Ras</i> gene is important for regulating the ERK activity, and hence expression of MMPs and uPA/uPAR. An activating mutation in the Ras gene can result in uncontrolled activity of MMPs and the urokinase system, which can lead to increased melanoma invasion. Blockade of PCs activity can reduce the expression and/or generation of active MMPs, uPA and uPAR leading to reduced tumor cells invasiveness. The asterisks indicate altered genes.</p

EMMPRIN up-regulates VEGFR-2 expression in primary melanoma cells.

<p><b>A</b>. M10 and WM278 cells were transfected for 24 hours with two different EMMPRIN siRNA (Ambion siRNA) or their corresponding scrambled control siRNA (Ctl siRNA) at 33 nmol/L concentration. EMMPRIN and VEGFR-2 protein expression was evaluated by Western blot analysis of 15 µg cell lysates respectively (actin was used as a loading control; representative blot of 3 independent experiments is shown) and conditioned medium (CM) was harvested for gelatinases and uPA zymographies. <b>B</b>. Immunofluorescence of M10 cells treated with EMMPRIN siRNA and immunostained for EMMPRIN, VEGFR-2, uPA and TIMP-1. <b>C</b>. QRT-PCR analyses of EMMPRIN, VEGFR-2 and TIMP-1 showing the mean ±SD of relative expression to <i>PPIA</i> house keeping gene of at least 3 independent experiments. <b>D</b>. M10 and WM278 cells were incubated with CHO-membranes containing or not EMMPRIN (Ctl and Emp respectively). VEGFR-2 and uPA transcript levels were quantified by qRT-PCR showing the mean ±SD of relative expression to <i>PPIA</i> house keeping gene of at least 3 independent experiments. VEGFR-2 protein expression was evaluated by Western blot analysis of 30 µg cell lysates (actin was used as a loading control; representative blot of 3 independent experiments is shown); bars, SD. *, p<0.05.</p

Furin, PACE4, PC5 and PC7 expression and activity in human primary melanoma cells.

<p>(<b>A</b>) Expression of the indicated PCs was analyzed in M10 cells using specific primers for the PCs found in the secretory pathway (Furin, PACE4, PC5, PC7) and reverse transcription-PCR analysis assay. Note that all these PCs are expressed in the M10 cells. (<b>B</b>) Following total RNA extraction from indicated cells, real-time PCR analysis was performed using specific primers for Furin, PACE4, PC5, PC7 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009992#s2" target="_blank">Materials and Methods</a>. During PCR, the transcription of β2-microglobulin that was evaluated in each sample was used as endogenous control. Results shown in the bar graph are expressed as ratio of PCs mRNA transcripts (M10)/(M10/PDX) deduced from values derived from M10 and M10/PDX cells mRNA analysis. Data are shown as means ± S.E of three experiments performed in duplicate. Real-time PCR analysis revealed that expression of α1-PDX in M10 cells did not affect significantly the expression levels of these PCs in M10 cells. Processing of proPDGF-A (<b>C</b>) and proIGF-IR (<b>D</b>) analyzed by Western blotting revealed that expression of α1-PDX in M10 cells (M10/PDX) completely inhibited the processing of pro-IGF-1R and proPDGF-A. (<b>E</b>) PCs activity in M10 cells and M10/PDX cells was assessed by evaluating the cell extracts for their ability to digest the universal PCs substrate, the fluorogenic peptide pERTKR-MCA at the indicated time points. Expression of α1-PDX in M10 cells reduced their PCs activity. (<b>F</b>) Results shown in the bar graph represent the PCs activity at 2 hours of incubation of the indicated tumor cells. Results are representative of three experiments and data are mean ± S.E performed in triplicate. ***p<i><</i>0.0001.</p

Expression of PC5 during fin regeneration.

<p>(A) Total RNA was isolated from fins (15-20 fins per time point) and analyzed by real-time PCR using specific primers for zebrafish PC5 or β-actin. Results are shown in the bar graph and are expressed as the ratio of the indicated transcripts relative to control (0 dpa). Results are shown as means ± S.E. of three experiments performed in triplicate. (B) Immunofluorescence analysis revealed that PC5 is expressed in all area of the regenerating fin and low signal was observed on the vessels (red signal, 25× objective).</p

Primers used for Real-time PCR analysis.

<p>Primers used for Real-time PCR analysis.</p