Research Paper Volume 1, Issue 10 pp 845—854

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).