after treatment with Dox 0.1 (magnification, 6200). (D)Western blot analysis of p16 INK4a evaluated 24 h after treatment with Dox 0.1 mM. (E) AV/PI staining evaluated 24 h after treatment with Dox 1 (magnification, 6200). doi:10.1371/journal.pone.0046126.g002 Doxorubicin Increases PPARd Protein Expression and Sequesters the Transcriptional Repressor Bcl6 in Unliganded PPARd, while L-165041 Treatment Increases Both PPARd and Free Bcl6 in H9c2 Cells PPARd mRNA (data not shown) and protein expression were significantly up-regulated in H9c2 cells treated with L-165041, doxorubicin 0.1 mM or exposed to sequential treatment with L- 165041 and doxorubicin 0.1 mM (Fig. 6). Since previous studies indicated that in the absence of a specific ligand, PPARd may bind Bcl6 [15,16], we thus examined the effects of doxorubicin and L- 165041 on Bcl6 and on PPARd:Bcl6 interactions by co- immunoprecipitation (Fig. 6A). Whole cell and nuclear extracts revealed that 0.1 mM doxorubicin did not change the expression levels of Bcl6 (Figs. 6A and 6C), but that it did cause a 2-fold increase in the amount of Bcl6 sequestered by PPARd (Fig. 6A). In contrast, while L-165041, either alone or followed by doxorubicin, increased total Bcl6 (Figs. 6A and 6C) it also decreased the amount of Bcl6 associated with PPARd (Fig. 6A) thus enhancing the amount of free Bcl6. By using specific inhibitors, we documented that p38, JNK and Akt play a key role in L-165041-induced Bcl6 up-regulation, and that Akt also regulates the L-165041-induced PPARd up-regulation. In fact, pre-treatment with the Akt inhibitor prevents the increase of PPARd protein levels in response to L- 165041 (Fig. 6C). These results suggest that L-165041 may counteract the action of doxorubicin through the increased expression and release of Bcl6. Bcl6 Plays a Key Role in the Regulation of Senescence in H9c2 The results we obtained prompted us to hypothesize that L- 165041 might counteract the action of doxorubicin through the increased expression and release of Bcl6. To better understand the influence of Bcl6 and Bcl6:PPARd interference on doxorubicin- Activated PPARd Protects from Senescence induced senescence, we selectively silenced either Bcl6 or PPARd using the siRNA transfection technique. We observed that the Bcl6 protein knock-down was associated with a significant increase in the number of SA-b-gal positive cells in both unstressed and 0.1 mM doxorubicin-treated cells, and that it completely abolished the anti-senescent effect of pre-treatment with the PPARd ligand L-165041(Figs. 7A, 7B and 7C). In contrast, silencing PPARd remarkably attenuated the pro-senescent effects of doxorubicin (Figs. 7A, 7B and 7C). Control siRNA, consisting of a pool of non- specific sequences, had no effect on SA-b-gal levels (Figs. 7A and 7C). We then became interested in assessing whether silencing Bcl6 could either cause apoptosis in untreated cells or produce a shift in the stress-response program from senescence to apoptosis in cells treated with doxorubicin 0.1 mM. Therefore, we examined the number of cleaved caspase-3-positive cells and we observed that the Bcl6 knock-down did not produce any effects in either untreated or in 0.1 mM doxorubicin-treated cells, with or without pre-treatment with the PPARd ligand L-165041 (Fig. 7D). Activated PPARd Inhibits Doxorubicin-induced Apoptosis In the previous paragraphs we reported data demonstrating that pre-treatment with the PPARd ligand L-165041 prevents senes- cence induced by doxorubicin 0.1 mM and that this effect mainly occurs through a Bcl6 related mechanism. We further examined the effects of pre-treatment with the PPARd ligand on cells exposed to pro-apoptotic doses of doxorubicin, and results show that pre-treatment with L-165041 prevents apoptosis induced by doxorubicin 1 mM, as assessed by both A/PI double staining (Fig. 2E) and cleaved caspase 3 (Fig. 8A). We found that doxorubicin 1 mM produces a two-fold increase Figure 3. Effects of L-165041 on the expression levels of PPAR isoforms. Effects of L-165041 on PPARa, PPARc and PPARd evaluated in neonatal cardiomyocytes 4 h and 22 h after L-165041 treatment. *p,0.05 versus time 0, 1p,0.05 versus 4 h. doi:10.1371/journal.pone.0046126.g003 Figure 4. Pre-treatment with p38, JNK and Akt inhibitors prevents the anti-senescent effects of L-165041. The specific inhibitors of p38 (SB203580, SB), JNK (SP600125, SP), Akt (Akt1/2 kinase inhibitor, Akti) but not the inhibitor of ERK1/2 (PD98059, PD) reverse the effects of L-165041 (L-165) on doxorubicin-induced SA-b-gal activity. *p,0.05 versus untreated cells, 1p,0.05 versus doxorubicin (Dox 0.1), # p,0.05 versus L-165+Dox 0.1. doi:10.1371/journal.pone.0046126.g004 PLOS ONE | www.plosone.org 5 September 2012 | Volume 7 | Issue 9 | e46126