Figure 3.Transcriptional activity of nutlin-induced p53 is attenuated in senescent WI-38 cells.
(A) Protein level of p53 and two target genes, p21 and MDM2, in
early passage and senescent cells. Cells were incubated in the presence of
10 μM nutlin-3a for
24 hours, lysed and subjected to Western blotting as described. (B)
Induction of p53 target genes by nutlin-3a is decreased in senescent cells.
Cells were treated as in (A), RNA was extracted and subjected to
quantitative PCR. Fold induction is calculated as change in post compared
to pre-treatment expression levels, both normalized to 18S rRNA. (C)
Total expression levels of p53 target genes are reduced in nutlin-3a
treated senescent cells. Cells were treated as in (B). Gene
expression levels after exposure to nutlin are shown normalized to
expression levels in early passage cells (100%). (D) Induction of
p53 regulated microRNAs by nutlin-3a is decreased in senescent cells. Cells
were treated as in (B). MicroRNA expression is determined by
quantitative PCR and normalized to RNU48. Fold induction is calculated as
in (B).(E) Total expression
levels of p53-regulated microRNA are reduced in nutlin-treated senescent
cells. Cells are treated as in (B), microRNA determined as in (D),
and data presented as in (C).
