Effects of p53 or AMPK knock-down and AMPK activation on p21 and p53 expression.

<p><b>a. Effects of p53-knockown on p53 and p21^CIP1expressions.</b> Cells grown on 6 well plates were infected with lentiviral vector, expressing short-hairpin (sh) RNA targeting non-human sequence (Control) or p53, at 30% confluence. Forty-eight hours later, cells were treated with 50 µM H₂O₂ for 2 hr, and harvested after 1 hr (for p53 expression) or 16 hours (p21^CIP1 expression). The knock-down of p53 completely blocked H₂O₂ induced upregulation of p53 and subsequent in p21^CIP1. Experiments were performed in duplicate and representative blots are shown. <b>b. Effects of AMPK knock-down on p53 and p21^CIP1 expression.</b> AMPK knock-down effects were assessed as in the preceding experiment. Cells were harvested at 16 hr after H₂O₂ treatment. AMPK knock-down increased both p53 and p21^CIP1 expression and H₂O₂ increased p21^CIP1 further. Experiments were performed in duplicate and a representative blots is shown. The result is consistent with the increased SA-Gal expression casued by AMPK knock-down as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035092#pone-0035092-g005" target="_blank">fig. 5</a>. <b>c, d. Effects of AICAR and resveratrol on H₂O₂ induced p21^CIP1 expression.</b> (<b>c.</b>) Cells grown on 6 well plates were treated with 50 µM H₂O₂ for 2 hr with/without 30 min pretreatment with 50 µM resveratrol or 1 mM AICAR. After sixteen hours of incubation, in standard growth medium, the cells were fixed with 2% paraformaldehyde and immunostained for p21^CIP1. H₂O₂ significantly increased positive staining in the nucleus (non-specific staining can be seen in cytosol), and this was largely prevented by AICAR and resveratrol. Scale bar = 50 µm. (<b>d.</b>) Quantitation of 150–200 cells from (a) revealed that approximately 50% of cells treated with hydrogen peroxide exhibited predominantly nuclear staining of p21^CIP1 and that AICAR and resveratrol completely prevented this from occurring. (n = 3, ** p<0.01). <b>e–f. Effects of AICAR on H₂O₂-induced p53 expression and phosphorylation.</b> (<b>e.</b>) p53 phosphorylation was increased by H₂O₂ exposure and this was not affected by AICAR (n = 3, ** p<0.01 vs 6). (<b>f.</b>) Cells were incubated with 1 mM AICAR for 1 hr prior to addition of 50 µM H2O2 and then harvested 1 hr later. p53 protein abundance was increased by H₂O₂ and AICAR had an additive effect on this. <b>e.</b> Increases in p53 abundance in H₂O₂ only and H₂O₂+AICAR groups observed at 1 hr were not observed at 16 hr. <b>g. p53 reporter gene assay.</b> The cells were infected with a p53-luciferase reporter adenovirus. Twenty-four hours later, one group was pre-treated for 1 hr with 1 mM AICAR then given 50 µH₂O₂. Six hours later the cells were harvested and luciferase activity assessed. Luciferase activity, which reflects p53 transactivation, was significantly increased by H₂O₂ and was suppressed by 1 mM AICAR. (n = 4, p<0.05 for both).