Furin, PACE4, PC5 and PC7 expression and activity in human primary melanoma cells.
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(A) 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) Following total RNA extraction from indicated cells, real-time PCR analysis was performed using specific primers for Furin, PACE4, PC5, PC7 as described in Materials and Methods. 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 (C) and proIGF-IR (D) 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. (E) 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. (F) 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<0.0001.