quantified by densitometry using an image analyzer system (Syngene, UK). Filters were probed with GAPDH in order to normalize the amounts of TRF2, PPARd and Bcldand with p38, JNK, Akt, ERK1/2 antibodies to normalize the amounts of pp38 and pJNK, pAkt, pERK1/2, respectively. The immunoblots of nuclear extracts were hybridized with anti-TATA binding protein antibody (Abcam) to monitor equivalent loading in different lanes. SA-b-gal Activity (Senescence-associated -b- Galactosidase Staining) Cells were stained for b-gal activity as described by Dimri [58]. The ability to induce SA-b-gal activity is a manifestation of residual lysosomal activity at suboptimal pH (pH 6). It becomes detectable in the course of senescence because of the increased lysosomal content on senescent cells [59]. The number of SA-b-gal positive cells was determined in 100 randomly chosen, low-power fields (x100) and expressed as a percentage of all counted cells. We chose to analyze the level of senescence three days after the end of the treatments because, had it been analyzed earlier, the percentage of senescent cells would have been too low to allow statistical analysis. MTT Assay The assay, which is based on the reduction of the tetrazolium salt MTT by active mitochondria to produce insoluble formazan salt, measures mitochondrial metabolic activity and is often used as an indicator of cell viability. Cells were treated in 96-well plates, MTT was added to each well under sterile conditions (final concentration of 5 mg/ml), and the plates were incubated for 3 h at 37uC. Formazan crystals were dissolved in dimethyl sulfoxide (100 ml/well). The purple formazan crystals were formed from yellow MTT by succinate dehydrogenase in viable cells. Absor- bance of the formazan product was measured at 570 nm with a background correction at 690 nm using a microplate reader [60]. Annexin V–fluorescein Isothiocyanate (FITC)/Propidium Iodide Staining Cells were labeled with AV/PI, and 100 randomly selected fields were counted using a fluorescence microscope. The number of stained cells was normalized to the total number of cells as counted by phase contrast microscopy of the same field. Immunocytochemistry Expression of cleaved caspase3 protein was documented by immunostaining using the Cleaved Caspase-3 (Asp175, Cell Signaling) antibody and was performed with the procedure described elsewhere [61]. This antibody detects endogenous levels of the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175. Caspase-3 is a critical executioner of apoptosis. Activation of caspase-3 requires proteo- lytic processing of its inactive zymogen into activated p17 and p12 fragments. Cells were examined by light microscopy for image analysis. References Activated PPARd Protects from Senescence Flow Cytometric Analysis Trypsinized and floating cells were pooled, washed twice with PBS and resuspended in 400 ml of hypotonic labeled solution, 5 mg/ml PI, 0.1% w/v Na citrate, 0.1%Triton X-100 in sterile water. Cells were incubated on ice for 30 min. until DNA content analysis. Both nuclear DNA content and cell cycle analysis were monitored by Fluorescence Activated Cell Sorting (Becton Dickinson, San Jose, CA) and data were analyzed using CellQuest software (Becton Dickinson San Jose, CA). F-actin Detection Cardiomyocytes growing on slides were fixed, permeabilized and labeled simultaneously in PBS containing 50 mg/ml lysopal- mitoyphospatidylecholine, 3.7% formaldehyde and 5 units/ml of fluorescent phallotoxin (A-12379alexaTM488 phalloidin, Molec- ular probes, Inc). Cells were rapidly washed three times with PBS and were viewed by fluorescent microscopy [62] in order to carry out image analysis. Bcl6 and PPARd siRNA Transfection ON-TARGET plus SMARTpool short interfering RNAs (siRNA) for silencing the expression of target genes Bcl6, PPARd and ON-TARGETplus Non-Targeting Pool as a control were purchased from Dharmacon (Thermo Ficher Scientific, USA). All transfections were carried out according to the manufacturer’s instructions with DharmaFECT1 transfection reagent (Thermo Fisher Scientific, USA). Briefly, cardiomyocytes were trypsinized, counted, and plated at a density of 104 cells/cm2. After 24 hours, cells were transfected with 100 nmol/l of SMARTpool siRNA or control siRNA using DharmaFECT1 reagent and analyzed after 24, 48 or 72 hours by immunocytochemistry for caspase 3, SA-b- gal activity and Western blot analysis. Image Analysis Image analysis was performed by the Leica Q500 MC Image Analysis System (Leica, Cambridge, UK). Three hundred cells were randomly analyzed for each sample, and the optical density of the signals was quantitated by a computer. The video image was generated by a CCD Camera connected through a frame grabber to a computer. Single images were digitized for image analysis at 256 grey levels. Imported data were quantitatively analyzed by Q500MC Software-Qwin (Leica, Cambridge, UK). The single cells were randomly selected by the operator by using the cursor and then positive areas were automatically estimated. Statistical Analysis Data are reported as mean6standard error of four independent experiments. Statistical analysis was performed by one-way ANOVA followed by the Bonferroni post-hoc test and by the Wilcoxon signed rank test when appropriate. Author Contributions Conceived and designed the experiments: PA PS C. Brunelli. Performed the experiments: PA C. Barisione SG AG PF. Analyzed the data: PA PS GG. Wrote the paper: PA PS. 1. Hershman DL, McBride RB, Eisenberger A, Tsai WY, Grann VR, et al. (2008) Doxorubicin, cardiac risk factors, and cardiac toxicity in elderly patients with diffuse B-cell non-Hodgkin’s lymphoma. J Clin Oncol 26: 3159–3165. 2. Auner HW, Tinchon C, Linkesch W, Halwachs-Baumann G, Sill H (2001) Correspondence re: O. J. Arola, et al., (2000) Acute doxorubicin cardiotoxicity involves cardiomyocyte apoptosis Cancer Res 60: 1789–1792. Cancer Res 61: 2335–2336. 3. Maejima Y, Adachi S, Ito H, Hirao K, Isobe M (2008) Induction of premature senescence in cardiomyocytes by doxorubicin as a novel mechanism of myocardial damage. Aging Cell 7: 125–136. PLOS ONE | www.plosone.org 12 September 2012 | Volume 7 | Issue 9 | e46126