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. 2013 Oct 1;5(11):1662–1683. doi: 10.1002/emmm.201202055

Figure 5. Oxygenation inhibits acetylation of p53-Lys118 residue and increases survival of cardiomyocytes.

Figure 5

  1. p53 knockout and p53Lys118–Ala118 mutant H9c2 cells were created as described previously (Gogna et al, 2012c). Western-blot analysis shows the knockout of p53 in H9c2 cells (lane 2), while addition of p53Lys118–Ala118 cDNA or p53 Wt cDNA to the H9c2 cells show the expression of p53 (lanes 3,4).
  2. The H9c2, H9c2 p53−/− and H9c2 p53-Lys118(Mut) cells were cultured under serum-deprived (SD) conditions to mimic MI. p53-Lys118 acetylation was analysed in the SD and oxygenated (Oxy) cells by Western blot (WB) and immunoprecipitation (IP) using p53-Lys118 antibody.
  3. The survival potential of H9c2, H9c2 p53−/−, H9c2 p53-Lys118(Mut) and H9c2 p53−/− +p53 Wt-cDNA cells was determined in the control, SD and SD + Oxy treatments. Data represent mean ± SD of eight independent measurements. *p = 6.6E−14 versus respective SD group; #p = 0.0014 versus respective SD group. The results show that oxygenation restored the SD-induced cell death.
  4. Results of luciferase assay showing the activation of p53-NOS3-RE and p53-BAX-RE in H9c2 cells. Oxygenation of SD cells switches from p53-BAX-RE to p53-NOS3-RE activation. Data represent mean ± SD of eight independent measurements.
  5. ChIP assay showing the binding of p53 to its respective NOS3 and BAX RE in H9c2 cells. The data show that p53 binds to BAX-RE in SD cells and shifts its binding to NOS3 upon oxygenation. No binding to either NOS3-RE or BAX-RE is observed in H9c2 p53−/− cells. In H9c2 p53-Lys118(Mut) cells, SD does not induce binding of p53 to the BAX-RE but upon oxygenation p53 binds to the NOS3-RE. The data suggest that p53-Lys118 acetylation is crucial for binding of p53 to BAX-RE and mutation in this site induces p53 binding to the NOS3-RE. Similar results of NOS3 and BAX mRNA and protein upregulation are observed in RT-PCR and the Western-blot assay.
  6. Expression of p53 downstream genes involved in apoptosis in H9c2 cells. The data show that SD results in the activation of these genes, whereas p53−/− and p53-Lys118 (Mut) cells do not activate these genes under any condition.