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-RASQ61K
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-RASQ61K or the copGFP
control.
(C)
The impact of pRb-silencing on the N-RASQ61K 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.
