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

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

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

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

Free IS levels, CD14⁺CD16⁺ monocytes and eGFR in study populations.

<p>(A) IS serum levels in AAA patients versus age- and e-GFR-matched control subjects, **p<0.01; (B) scattergram and regression line representing the relationship of CD14⁺CD16⁺monocyte percentages with IS serum levels (p = 0,014; r = 0,34); (C) and (D): scattergram and regression line representing the relationship between IS concentrations and eGFR in control subjects (empty circle, p = 0,02; r = -0,55) and AAA patients (grey triangle, p = 0,2; r = -0,17) respectively.</p

Proposed scheme of IS effects on monocyte differentiation.

<p>Mild IS concentrations (1, 10, 20 μM) potentiate the detoxifying and anti-oxidant pathways of AhR and Nrf2 in monocytes (THP-1 cells), resulting in CD163 and HO1 overexpression and monocyte activation. Macrophages derived from IS-treated monocytes (IS-treated MdM) retain an upregulation of the AhR activity and features of a profibrotic, low inflammatory phenotype.</p

Gene expression in IS-treated M/2M.

<p>RT-PCR for (A) AhR, (B) AhRR, (C) Nrf2, (D) HO1, (E) IL-6 and (F) CCL2. Results were plotted as mRNA fold induction in IS-treated versus Control cells; * p <0.05; ** p <0.01; ***p<0,001 vs. control cells.</p

Genomic spectrum of acquired driver alterations.

A) The circle graph represents for each case (n = 74) the proportion of driver mutations detected in primary and/or metastatic tumor samples. Outer numbers represent mutations of eBC, inner numbers represent mutations of mBC. B) Cumulative frequency of the difference (Δ) between number of mutations in metastatic vs. primary tumor samples (Δ vs. metastatic tumor; Δ > 0, number of driver mutations in the primary sample lower than in the metastatic sample. C) Non-linear relationship between the difference of driver mutations in metastasis/primary pair (Δ, x-axis), and DRFS hazard ratio of Schoenfeld residuals (y-axis). The analysis is adjusted for T/N status, Ki67, menopausal status and tumor grade. The solid line represents a penalized spline fit of the predicting variables, while the dashed lines show 95% confidence intervals. D) Functional analysis of Gene Ontology (GO) terms associated to cell cycle, DDR, epigenetic regulation, androgen receptor activity and WNT signaling pathway. The size of the dots is inversely proportional to the p values of estimated hazard ratio (x-axis) displayed in log10 scale. P values are reported in S5 Table.</p