Figure 3

Cells overexpressing EphA2 or RacN17, treated with Rho activator or ilomastat promote MAT and induce an increase in stem cell markers and clonogenic potential. A-B) Hs294T, EphA2 or RacN17 transfected cells were serum starved for 48 h, for Ilomastat treatment cells were serum starved for 48 h in the presence of 50 μmol/L Ilomastat, for Rho activation treatment cells were serum starved for 48 h and then stimulated with 1 U/ml Calpeptin for 2 h at 37°C. Cells were then analysed for expression of the cell-surface markers CD20 and CD133 by means of cytometry. The CD20 or and CD133-positive populations were plotted. The results are representative of three experiments with similar results. Student t-test, *p < 0.001 treatments vs control. C-F) Cells were treated as in A). Total RNA was extracted and NANOG, KLF4, SOX2 and OCT4 mRNA expression level was analyzed by qRT-PCR. Results are representative of three experiments with similar results. Student t-test, *p < 0.005 treatments vs control. G) Representative images of clones obtained from cells treated as in A) after 10 days of culturing at clonal densities. Bar, 100 μm. Clones were photographed, counted and the mean ± SD of clones number/field is reported. H) P1 individual spheres, derived from dissociated single melanospheres, were counted and the bar graphs obtained from triplicate experiments are shown. Student t-test, *p < 0.005.