Activated PPARd Protects from Senescence Figure 6. L-165041 and doxorubicin regulate PPARd and Bcl6 expression and interaction: the role of MAPKs and Akt. H9c2 cells were pre-treated with or without L-165041 (L-165) for 2 h, then treated with or without doxorubicin (Dox) 0.1 mM for 3 h and analyzed after 24 h. Dox 0.1 increases PPARd levels and enhances the interaction between Bcl6 and PPARd but does not modify the amount of Bcl6. L-165041 increases the protein levels of both PPARd and Bcl6 and induces the release of Bcl6 from PPARd. Cytoplasmic extracts reveal nuclear localization of PPARd and Bcl6. (A) Nuclear extracts were assayed by western blot analysis with PPARd or Bcl6 antibodies; co-immunoprecipitation of PPARd and Bcl6 (IP-PPARd WB- Bcl6) was performed with anti- PPARd followed by western blot using the anti-Bcl6 antibody. The bar graph shows the protein quantification expressed as a percentage. *p,0.05 versus control (ct), 1p,0.05 versus Dox 0.1, # p,0.05 versus L-165141+Dox 0.1. (B) Cytoplasmic extracts were evaluated by western blot with PPARd and Bcl6 antibodies. (C) The effects of SB203580 (SB), inhibitor of p38, SP600125 (SP), inhibitor of JNK and Akt1/2 kinase inhibitor (Akti), inhibitor of Akt, on the L-165041-induced increases of PPARd (first line) and Bcl6 (second line). Whole cell extracts were assayed by western blot analysis. All inhibitors reverse the effects of L-165041 on Bcl6 levels, while only the Akt inhibitor reverses the up-regulation of PPARd. The bar graph shows the protein quantification expressed as a percentage. *p,0.05 versus ct, 1p,0.05 versus Dox 0.1, # p,0.05 versus L- 165141. doi:10.1371/journal.pone.0046126.g006 PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e46126