Bcl6 plays a key role in cellular senescence.

<p>Bcl6 siRNA(siBcl6) significantly increases the number of SA-b-gal positive cells in both unstressed and in 0.1 µM doxorubicin-treated cells, and it completely abolishes the anti-senescent effect of pre-treatment with L-165041 (L-165). siPPARδ completely abolishes the pro-senescent effect of 0.1 µM doxorubicin. H9c2 cells were transfected with Bcl6-, PPARδ specific or control (siCT) siRNA for 48 h before treatment with or without L-165041 for 2 h followed by treatment with or without 0.1 µM doxorubicin (Dox 0.1) for 3 h. Cells were assayed for protein amount (after 24 h), premature senescence (after 3 days) and apoptosis (after 24 h). (A) Western blot analysis with Bcl6 and PPARδ antibodies. (B) SA-b-gal activity (magnification ×200). (C) Bar graph illustrating the percentage of SA-b-gal positive cells. *<i>p</i><0.05 versus corresponding siCT of the same treatment condition, (D) Bar graph illustrating the percentage of caspase-3 positive cells.</p

Pre-treatment with p38, JNK and Akt inhibitors prevents the anti-senescent effects of L-165041.

<p>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. *<i>p</i><0.05 versus untreated cells, §<i>p</i><0.05 versus doxorubicin (Dox 0.1), # <i>p</i><0.05 versus L-165+Dox 0.1.</p

Effects of L-165041 and/or doxorubicin 0.1 µM on MAPKs and Akt phosphorylation.

<p>Both L-165041(L-165) and doxorubicin (Dox) 0.1 µM activate MAPKs and Akt. If cells are pre-treated with L-165, the doxorubicin-induced increase of pJNK and pAkt levels is inhibited, pERK levels are maintained sustained, while pp38 levels result higher than those induced by Dox 0.1 alone. (A) Time curve analysis of phosphorylated pp38, pJNK, pAKT, pERK1/2 and total p38, JNK, AKT, ERK1/2 evaluated by western blot. (B, C, D, E) Graphs showing values for pp38, pJNK, pAKT and pERK1/2 normalized to the amount of total enzyme.</p

Effects of L-165041 on the expression levels of PPAR isoforms.

<p>Effects of L-165041 on PPARα, PPARγ and PPARδ evaluated in neonatal cardiomyocytes 4 h and 22 h after L-165041 treatment. *<i>p</i><0.05 versus time 0, §<i>p</i><0.05 versus 4 h.</p

L-165041 prevents both senescence and apoptosis induced by doxorubicin.

<p>Pre-treatment with the PPARδ agonist L-165041 (L-165) prevents both senescence induced by low (0.1 µM) doses of doxorubicin (Dox) (A,B,C), and apoptosis induced by high (1 µM) doses of Dox (E) in neonatal rat cardiomyocytes. (A) Percentage of SA-b-gal positive cells 3 days after treatment. *<i>p</i><0.05 versus control (ct), §<i>p</i><0.05 versus Dox 0.1. (B) Cell size, 3 and 5 days after treatment. *<i>p</i><0.05 versus ct, §<i>p</i><0.05 versus Dox 0.1. (C) Photographs showing cells (from top to bottom): SA-b-gal activity evaluated 3 days after treatment with Dox 0.1 (magnification, ×200), F-actin density (magnification, ×400), and AV/PI staining evaluated 24 h after treatment with Dox 0.1 (magnification, ×200). (D)Western blot analysis of p16 INK4a evaluated 24 h after treatment with Dox 0.1 µM. (E) AV/PI staining evaluated 24 h after treatment with Dox 1 (magnification, ×200).</p

L-165041 protects H9c2 from doxorubicin-induced apoptosis through a Bcl6-independent mechanism.

<p>(A) Pre-treatment with L-165141decreases the number of doxorubicin-induced apoptotic cells. Bcl6 siRNA (siBcl6) does not modify the number of activated caspase-3 positive cells in any of the treatment groups i.e., untreated cells, 1 µM doxorubicin-treated cells, and in cells pretreated with L-165041 and then incubated with doxorubicin. 24 h after treatment, cells were assayed by immunocytochemistry for cleaved caspase-3. Bar graph illustrates the percentage of cleaved-caspase-3 positive cells. *<i>P</i><0.05 versus ct, §<i>P</i><0.05 versus doxorubicin. (B) Effects of L-165041(L-165) and 1 µM doxorubicin (Dox1) on PPARδ and Bcl6 protein expression and on PPARδ and Bcl6 protein interaction. Doxorubicin increases PPARδ levels and down-regulates the amount of total and PPARδ bound Bcl6 (IP-PPARδ WB-Bcl6). Pre-treatment with L-165041 does not modify the effects induced by doxorubicin. H9c2 cells were pre-treated with or without L-165041 for 2 h, then treated with or without 1 µM doxorubicin for 3 h and analyzed after 24 h by western blot. The bar graph shows the protein quantification expressed as a percentage. *<i>p</i><0.05 versus control (ct).</p

Phalloidin staining of F-actin.

<p>(A) Photographs depict EPCs pre-treated with or without MAPK inhibitors, and then treated with 0.25 µM doxorubicin (magnification 1000X). (B) Bar-graph showing cell size in various treatment groups. ct, control; Dox, 0.25 µM doxorubicin. SB, p38 inhibitor; SP, JNK inhibitor. *p<0.05 vs ct; § p<0.05 vs Dox.</p

Doxorubicin induces cell cycle alterations and impairs cell viability.

<p>(A) Cell cycle analysis: the blue area represents the control cells, the red line indicates treated cells. Cells were cultured for 24 h after treatment with doxorubicin 0,25 µM and then analyzed. Phases of the cell cycle are indicated: Sub G0 [M1], G0/G1 [M2], S [M3], G2/M [M4], >4N [M5]. (B) Cell proliferation evaluated by BrdU incorporation. Cells were cultured for 24 h after treatment with various doses of doxorubicin and then analyzed. BrdU data were normalized to untreated (media alone) cells (C), Number of viable cells evaluated by MTT assay. Cells were analyzed before treatment and 24 hours after treatment with various doses doxorubicin. ct, control; Dox, doxorubicin. *p<0.05 vs ct. § p<0.05 vs 0 h; # p<0.05 vs ct 24 hr.</p

EPC migration assay.

<p>Graph represents the percentage of migrating EPCs assessed by a modified Boyden chamber assay; EPCs were untreated or incubated with 0.25 µM doxorubicin and then exposed to: VEGF, medium, or conditioned medium from normal or doxorubin-induced senescent or apoptotic H9c2 cells. ct, control; Dox, doxorubicin; SP, JNK inhibitor; SB, p38 inhibitor; ct, control; *p<0.05 vs medium; # p<0.05 vs untreated cells exposed to VEGF; ¤ p<0.05 vs. all conditions.</p

Dose-effect curve.

<p>(A) Bar-graph showing the percentages of SA-b-gal and ssDNA positive cells after treatment with various doses of doxorubicin. (B) Photographs illustrating the effects of doxorubicin 0.25 and 1 µM. Cells were evaluated for (from top to bottom): SA-b-gal activity (magnification, ×200), ss-DNA positivity (magnification, ×200), and AV/PI staining (magnification, ×400). ct, control; Dox, doxorubicin. *p<0.05 vs control.</p