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

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© 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO

Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation.

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Western-blot results show the abolition of p53 expression in RNC cells (lane 2), while addition of p53^{Lys118-Ala118} cDNA and p53 Wt cDNA to RNC p53^−/− cells show the expression of p53 (lanes 3 and 4). Control cells show expression of p53 (lane 1).

Immunoprecipitation results show acetylation of p53 at ^Lys118 residue in RNC cells under SD conditions (lane 2). However, this expression is abolished upon oxygenation (lane 3). Acetylation at p53 ^Lys118 is absent in control, RNC p53^−/− and RNC p53-Lys¹¹⁸(Mut) cells under both SD and SD + Oxy conditions (lanes 4–9).

Cell survival analysis of control, SD and SD + Oxy-treated RNC, RNC + p53 siRNA, RNC + p53 siRNA + p53-Lys¹¹⁸(Mut) cDNA and RNC + p53 siRNA + Wt p53 cDNA cells show an increase in the SD-treated RNC + p53 siRNA and RNC + p53 siRNA + p53-Lys¹¹⁸(Mut) cDNA cells compared to RNC cells, exhibiting the crucial role of deacetylation of p53 Lys¹¹⁸ in cell survival. Data represent mean ± SD of eight independent measurements. *p = 2.6E−14 versus respective SD group; ^#p = 9.3E−08 versus respective SD group.

Luciferase assay shows activation of BAX-p53-RE and NOS3-p53-RE in SD and SD + Oxy-treated RNC cells. Neither BAX nor NOS3 is activated in RNC + p53 siRNA cells. Oxygenation caused a switch of BAX activation to NOS3 activation in RNC p53-Lys¹¹⁸(Mut) cells. Data represent mean ± SD of eight independent measurements.

ChIP assay shows the binding of p53 to its respective NOS3 and BAX RE in RNC cells (lanes 4 and 5). However, no binding of p53 to BAX-RE or NOS3-RE is observed in RNC p53^−/− cells (lanes 7 and 8). However, p53 binds to NOS3-RE in SD + Oxy-treated RNC p53-Lys¹¹⁸(Mut) cells (lane 11). These results suggest the importance of p53 ^Lys118 acetylation in activation of either BAX-RE or NOS3-RE and regulation of apoptotic or survival pathway respectively. Similar results were observed at mRNA and protein level using RT-PCR and Western-blot techniques in RNC, RNC p53^−/− and RNC p53-Lys¹¹⁸(Mut) cells.

Expression of p53 downstream genes in RNC, RNC p53^−/− and RNC p53-Lys¹¹⁸(Mut) cells. SD-treated RNC cells show activation of apoptotic genes whereas RNC p53^−/− and RNC p53-Lys¹¹⁸(Mut) cells show no involvement in apoptotic gene activation.