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.