Figure 5

Only senescent NRAS^61K cells give rise to anoikis-resistant progeny. (a) Melan-a control cells (upper images) and N-RAS^61K cells (lower images) were cultivated in the presence of doxycycline (Dox) before they were transiently transfected with the pGreenFire-p53 reporter plasmid. Note the strong GFP staining for the multinucleated N-RAS^61K melanocytes. Scale bars, 75 μm. (b) After 1 week of doxycycline stimulation, p53-GFP-transfected N-RAS^61K cells were sorted for large cell size (as characteristic for the senescent multinucleated cells) and strong GFP positivity. (c) Appearance of cells 2 weeks after sorting (left) and 1 week later (right). Scale bars, 35 μm. (d) After 6 weeks, supernatant from sorted cells was transferred to a new dish and was allowed to attach overnight, before a crystal violet staining was performed. Please note that due to the low initial cell density in the dishes, the amount of cells in the supernatant is much lower than in previous figures, thus giving only rise to weak crystal violet staining. Arrows indicate the presence of some exemplary cell colonies (left). A magnification shows the accumulation of many cells to one colony, suggesting that the cells that gave rise to the colony were present as spheroid in the cell culture supernatant (right). Scale bar, 50 μm. (e) Proliferation of melan-a (ctrl.), and unsorted N-RAS^61K cells (N-RAS^61K) as well as N-RAS^61K cells sorted for GFP and large cell size (N-RAS^61K-s) and corresponding anoikis-resistant cells from the supernatant (N-RAS^61K-s-AR). Cells were either kept in starving medium (10% dialyzed FCS) containing Dox or in normal culture medium without TPA, as indicated. Asterisks indicate statistical significance of N-RAS^61K-s and N-RAS^61K-s-AR cells compared with N-RAS^61K cells. *P<0.05; **P<0.01, ***P<0.001; n=3. (f) Soft agar growth assay of N-RAS^61K, N-RAS^61K-s and N-RAS^61K-s-AR cells, cultivated for 14 days in DMEM containing 10% FCS and antibiotics