Research Paper Volume 1, Issue 6 pp 542—556

Figure 2. Oncogenic N-RAS ^Q61K induces DNA damage response in human melanocytes. (A) Human epidermal melanocytes infected with lentiviruses expressing N-RAS^Q61K or copGFP were stained with DAPI and antibodies to the DNA damage marker γ-H2AX, 15 days post transduction (bar =10 μm). (B) Human melanocytes were transduced with lentiviruses expressing N-RAS^Q61K or copGFP and cultured for 15 days in the presence (+) or absence (-) of 4mM caffeine. Expression of the indicated proteins was determined by western blot analysis 15 days after infection. (C) Melanocytes transduced with lentivirus expressing N-RAS^Q61K or copGFP and cultured for 15 days in the presence (+) or absence (-) of 4mM caffeine were stained with DAPI and antibodies against the phosphorylated forms of p53 (p-p53) or CHK2 (p-CHK2) (bar=100μm). Enlarged images of representative cells (marked with arrow) are also shown. The percentage of transduced melanocytes positive for p-p53 and p-CHK2 expression was quantitated from at least two independent transduction experiments from a total of at least 300 cells. The graph corresponds to the mean percentage of transduced cells treated with caffeine (+) or left untreated (-) ± s.d. (D) Human melanocytes were transduced with lentiviruses expressing N-RAS^Q61K or copGFP and cultured for 15 days in presence (+) or absence (-) of 4mM caffeine. The efficiency of transduction was controlled with the co-expression of copGFP and was consistently above 90%. Cell proliferation (Ki67), chromatin condensation (DAPI), and the appearance of increased SA-β-Gal activity were analyzed and quantitated 15 days after infection. Percentage of cells positive for the indicated marker is shown in histograms, which correspond to the mean ± s.d. of at least two independent transduction experiments from a total of at least 300 cells. Cells enlarged to show DAPI-stained chromatin foci are indicated with arrows (bar =10 μm). LM, light microscopy (bar=100μm).