Research Paper Volume 1, Issue 6 pp 542—556

Figure 3. Relative contributions of the p53 and pRb tumour suppressor pathways in N-RAS ^Q61K-induced melanocyte senescence. (A) Melanocytes were transduced with lentiviruses containing the indicated shRNA constructs. Three days post infection the cells were re-transduced with lentiviruses expressing N-RAS^Q61K or copGFP, as shown. Representative examples at 15days after infection are shown. Cell proliferation (Ki67), chromatin condensation (DAPI), and the appearance of increased SA-β-Gal activity were analyzed and quantitated. Percentage of cells positive for each indicated marker are 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). (B) Expression of the indicated proteins was determined by western blot analysis at 15 days after infection of human epidermal melanocytes with the indicated shRNA constructs and either lentivirus expressing N-RAS^Q61K or the copGFP control. (C) The impact of pRb-silencing on the N-RAS^Q61K induced senescence was determined by quantitating key senescence markers (Ki67 expression, SAHF formation, SA-β-Gal activity) at 10 and 15 days post N-RAS transduction. Percentage of cells positive for each 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.