Figure 5: The EPE peptide leads to apoptosis of B-Raf melanomas.

(a) Effect of the EPE peptide on the morphology of A2352, LOXIMVI, HeLa and NHEM-Ad melanoma cells. The indicated cells were seeded at ~25% confluence, and treated either with EPE or scrambled peptides (10 μM), MEK inhibitors (10 μM U0126 for the first 3, and 2 μM PD-184352 for NHEM-Ad) or DMSO control (0.1%), for 48 h. Images were obtained by light microscope after 72 h. Scale bar, 50 μm. (b) Effect of the EPE peptide on the cell apoptosis determined by TUNEL staining. LOXIMVI, A2352, NHEM-Ad or HeLa cell lines were treated either with EPE or scrambled peptides (10 μM), as well as positive (pos.) control (cont.; 30 nM Taxol for LOXIMVI, A2352 and HeLa cells; 100 μM H₂O₂, 3 h, for NHEM-Ad) or DMSO control (0.1%) for 24 h. TUNEL staining was performed according to manufacturer’s instruction (Roche). Images were obtained and analysed by florescence microscope. Scale bar, 50 μm. The bar graph represents per cent of apoptotic cells calculated in 10 different microscopic fields. (c) Effect of the EPE peptide on cell apoptosis determined by PARP cleavage. A2352, A2185, LOXIMVI, CHO (Chinese hamster ovary), HB2, MDA-MB or HeLa cells were treated as described above, and incubated for 24 h. Cell extracts were collected and 25 μg of protein were separated by 10% SDS–PAGE. The blots were subjected to an anti-cleaved PARP Ab. The closest molecular weight marker (kDa) is shown on the right of the relevant panels. All experiments were reproduced three times.