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

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

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

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

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

Effect of exogenous IGF-1 on doxorubicin-induced apoptosis of H9c2 cells: TUNEL and caspase 3/7 activity.

<p>Frequency of apoptotic cells, as assessed by TUNEL (A; representative microphotographs are shown in B) and fluorescence (AUF) produced by the cleavage of a substrate of activated caspase 3/7 (C), 24 hours after no treatment (Ctr) or incubation of H9c2 cardiomyocytes with doxorubicin (Dox) ± IGF-1 at the indicated concentrations. ***, P <0.001 vs. Ctr. c, P <0.001 vs. Dox 0.1; d, P <0.001 vs. Dox 0.5; e, P <0.01 vs. Dox 0.1; f, P <0.05 vs. Dox 0.5. ¥, P <0.001 vs. Dox 0.1 + IGF-1 100; ¢, P <0.001 vs. Dox 0.1 + IGF-1 100 and Dox 0.5 + IGF-1 100.</p

Antioxidants reverse doxorubicin-initiated apoptosis and IGF-1R /IGFBP-3 perturbation.

<p>Frequency of apoptotic cells (A) and IGF-1R and IGFBP-3 expression (representative western blot in B and densitometry of western blot bands in C) 24 hours after no treatment (Ctr) or incubation of H9c2 cardiomyocytes with 1 μM doxorubicin (Dox) with or without pre-treatment with N-acetylcysteine (NAC), dexrazoxane (DEX), or carvedilol (CARV). *, P <0.05 vs. Ctr; **, P <0.01 vs. Ctr; ***, P <0.001 vs. Ctr; ****, P <0.0001 vs. Ctr. #, P <0.05 vs. Dox 1; ^, P <0.01 vs. Dox 1.</p

Doxorubicin stimulates apoptosis and modulates IGF-1R/IGFBP-3 expression in H9c2 cells.

<p>Frequency of apoptotic cells (A) and IGF-1R (B) and IGFBP-3 (C) expression (densitometry of western blot bands) 24 hours after no treatment (Ctr) or incubation of H9c2 cardiomyocytes with 0.1, 0.5, or 1 μM doxorubicin (Dox). A representative western blot for IGF-1R and IGFBP-3 is shown in (D). *, P <0.05 vs. Ctr; **, P <0.01 vs. Ctr; ***, P <0.001 vs. Ctr. a, P <0.01 vs. Dox 0.5; b, P <0.05 vs. Dox 0.1; c, P <0.001 vs. Dox 0.1.</p

Involvement of p53 in the change in IGF-1R /IGFBP-3 levels caused by doxorubicin.

<p>(A) Representative western blot and band densitometry for p53 24 hours after no treatment (Ctr) or incubation of H9c2 cardiomyocytes with 0.1, 0.5, or 1 μM doxorubicin (Dox). (B and C) IGF-1R/IGFBP-3 expression (band densitometry and representative western blot, B) and annexin V/propidium iodide positivity (C) in H9c2 cardiomyocytes untreated or exposed to 1 μM Dox with or without pre-treatment with PFT-α. *, P <0.05 vs. Ctr; ***, P <0.001 vs. Ctr. ^, P <0.01 vs. Dox 1.</p