Figure 7.
Senescence in p63-ablated keratinocytes is prevented by knock-down of p53 and PML, but not by p16INK4a knock-down or ectopic DNp63α overexpression. (A) Real-time PCR analysis for the DBD, TA, and N-terminal truncated (DN) transcripts in response to Cre-mediated ablation in primary keratinocytes. (B) Western blot analysis for p63 in GFP-infected (V), Cre-infected (C), or DNp63α-infected (DN) keratinocytes, showing that the DNp63α isoform (arrow) and other putative p63 isoforms (dashed arrows) are efficiently ablated in Cre-infected cultures. Asterisk denotes nonspecific band. (C, top right) Real-time PCR analysis for transcripts encoding different p63 isoform subtypes in keratinocytes infected with GFP, DNp63α, or Cre + DNp63α. (Left) Phenotypic and fluorescent analyses of primary keratinocyte cultures infected with GFP- or Cre-expressing vectors, or with Cre in combination with DNp63α, or with Cre in combination with short hairpins specific for p53, p16INK4a, or PML. The proliferation defect was prevented in cultures treated with sh p53 and sh PML, but not sh p16 or DNp63α. (Bottom right) Cultures were assayed for SA-β-gal activity following infection, and the percentage of positive cells was quantitated. Bars, 100 μm.