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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 analysis was conducted to study the expression of TIP60 in control, SD and SD + Oxy-treated cells. SD induces TIP60 expression (lane 2); however, this expression is significantly inhibited by oxygenation (lane 3).

Western blots of TIP60 expression in MI and MI + Oxy-treated cardiac tissue. Results show increased TIP60 expression in MI tissue (lane 2); however, this expression is significantly reduced in MI + Oxy-treated cardiac tissue (lane 3).

Co-immunoprecipitation study was conducted to study p53 and TIP60 interaction in MI and MI + Oxy-treated RNC cells. Results show p53 interaction with TIP60 in SD-treated cells (lane 4); however, this oxygenation significantly inhibits this interaction (lane 5) in RNC cells. Input and TIP60 siRNA were used as controls for the study.

The role of TIP60 in cell survival was studied in RNC cells using flow cytometry. Transfection with TIP60 siRNA results in increase in cell-survival fraction in RNC cells while TIP60 cDNA results in a decrease in cell-survival fraction. Conversely, neither TIP60 siRNA nor TIP60 cDNA has any effect on cell survival in RNC p53^−/− and RNC p53-Lys¹¹⁸(Mut) cells. Data represent mean ± SD of four independent measurements.

CHIP assay was conducted to study the role of TIP60 in p53 binding to BAX-RE or NOS3-RE in SD and SD + Oxy-treated RNC cells. Results show that transfection with TIP60 siRNA results in p53 binding to NOS-RE in both SD and SD + Oxy-treated cells. However, exogenous addition of TIP60 cDNA result in p53 binding to BAX-RE in both SDF and SD + Oxy-treated cells.

Effect of TIP60 siRNA and TIP60 cDNA addition on p53-Lys¹¹⁸ acetylation, NOS3 and BAX protein expression was studied in SD and SD + Oxy-treated RNC cells. Transfection with TIP60 siRNA led to abolition of p53-Lys¹¹⁸ acetylation, increased NOS3 expression and decreased BAX expression in RNC cells. Contrarily, transfection with TIP60 cDNA led to increased p53-Lys¹¹⁸ acetylation, decreased NOS3 expression and increased BAX expression in RNC cells.

Gene array was studied to study the role of TIP60 in activation of p53 downstream genes in SD and SD + Oxy treated cells. Transfection with TIP60 cDNA results in the activations of apoptotic genes in SD and SD + Oxy-treated cells when compared to TIP60 siRNA-transfected SD and SD + Oxy-treated groups.