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. 2002 Jul;22(14):5157–5172. doi: 10.1128/MCB.22.14.5157-5172.2002

FIG. 3.

FIG. 3.

Expression and activity of cdk4R and p53DD in keratinocytes. (a) Western blots detecting expression of p53DD, p53, cdk4, the mutant cdk4R, and nuclear lamins A and C (as loading controls). Lane 1, strain N (34 PD); lane 2, strain N (44 PD); lane 3, N/p53DD (53 PD); lane 4, N/cdk4R/p53DD (69 PD); lane 5, N/cdk4R (46 PD); lane 6, POE9n (40 PD). Note elevated levels of endogenous p53 protein in p53DD-expressing cells and mutant cdk4R protein at severalfold higher levels than endogenous cdk4 protein in cdk4R-expressing cells. (b) cdk4R function in transductants. N/cdk4R cells at 54 PD (near senescence) treated with BUdR 24 h before fixation and stained immunocytochemically for BUdR (red) and p16 (blue). Note S-phase entry (BUdR incorporation) in cells containing high levels of p16. (c) Expression of p53DD in keratinocytes prevents growth arrest in response to DNA damage. Panels 1 and 2, strain N. Panels 3 and 4, N/p53DD. Cells shown in panels 2 and 4 were treated with 0.5 nM actinomycin D for the final 2 days before fixation. Cells shown in all panels were treated with 1 nM BUdR for the final 20 h before fixation. In each panel, the upper square shows BUdR immunocytochemical staining and the lower square is the phase contrast image of the same field. Note absence of normal growth arrest response in N/p53DD cells.