1 SUPPLEMENTAL INFORMATION - TABLE OF CONTENT 1. SUPPLEMENTALFIGURES AND LEGENDS.................................................................. 2 Figure S1. Purity checkon the nuclear and cytoplasmic fractions .................................................2 Figure S2. Diagrammaticrepresentation of pNOS3p-luc1vectorconstruct .................................. 3 Figure S3. Effectof p53 on NOS3 promoter...................................................................................4 Figure S4. Role of p53 in theupregulation of NOS3 gene inL6cells ..............................................5 Figure S5. Diagrammaticrepresentation of theminimal pNOS3p-luc2 vector construct ..............6 Figure S6. Co-localization ofp53 and NOS3 proteins inthe infarct hearttissue ...........................7 Figure S7. Binding of p53proteinon theBAX genepromoter .......................................................8 Figure S8. BAX mRNAlevel inthe infarct hearttissue .................................................................... 9 Figure S9. BAX protein level in the infarcthearttissue ................................................................ 10 Figure S10. Invivo ELISAof p53modifications .............................................................................11 2. SUPPLEMENTALEXPERIMENTALPROCEDURES....................................................... 12 Westernblotting...........................................................................................................................12 Preparation of nuclearand cytoplasmic extracts ......................................................................... 12 Immunoprecipitation ...................................................................................................................12 Annexin-V staining........................................................................................................................13 Protein ELISA ................................................................................................................................ 13 Induction ofmyocardialinfarction ...............................................................................................13 Protocol for oxygen-cycling .......................................................................................................... 14 Immunohistochemical staining of cardiac tissuefor p53 andnos3 expression ...........................14 2 1. SUPPLEMENTALFIGURES AND LEGENDS FigureS1. Purity check on thenuclear and cytoplasmic fractions.Immunoprecipitation (IP) with anti- PARP (nuclear protein) and anti-tubulin (cytoplasmic protein) antibodies was conducted in control (healthy) heart tissues to check the purity of the nuclear (NF) and cytoplasmic (CF) fractions. The images (representative of n=4) show that the nuclear and cytoplasmic fractions are pure as evidenced by the lackof tubulin proteininthe nuclearfraction andlackof PARP in the cytoplasmicfraction. 3 FigureS2. Diagrammatic representation of pNOS3p-luc1vector construct.The 602 base-pair NOS3 promoter,upstreamof the transcription startsite is shown. 4 FigureS3. Effect of p53 on NOS3 promoter. pNOS3p-luc1 was transfected into H4TG hepatoma cells. Resveratrol was used to activate p53 transcription, which induced a 12-fold increasein pNOS3p-luc1 in resveratrol-treated H4TG cells. p53 gene-silencing using p53 siRNA abolished the activation of p53 promoter. Datarepresent mean±SD of 8independent measurements. *p=2.1E-09. 5 FigureS4. Roleof p53 in theupregulation of NOS3 genein L6 cells. The cells were treated with 10- μM resveratrol to activate p53. Results show a marked increase in NOS3 mRNA and protein expression in the resveratrol-treated cells. Upon p53 gene-silencing using p53 siRNA, the resveratrol- mediated increase in NOS3 gene was abolished, suggesting a role for NOS3 in regulation of NOS3 gene. 6 FigureS5. Diagrammatic representationof theminimal pNOS3p-luc2 vector construct. pNOS3p-luc2 (NOS3-p53RE) andthe mmpNOS3p-luc2 (mutant minimal promoter2,with mutated p53RE sequence) vector constructs areshown. 7 FigureS6. Co-localization of p53 and NOS3 proteins in theinfarct heart tissue. Immunohistochemical staining of NOS3 and p53 were performed using NOS3 and p53 antibodies. The fluorescence microscopic images of DAPI (nuclear staining), p53 and NOS3 are superimposed to show their co- localization in the healthy (control), infarct (MI) and oxygen-treated(MI+OxCy)hearts. 8 FigureS7.Bindingofp53 proteinontheBAX genepromoter. Chromatinimmunoprecipitation (ChIP) was used to analyze thebinding of p53 protein on the BAX gene promoter in the MI and MI+OxCy treated cardiac tissue. Results show that p53 does not bind to the BAX promoter in the healthy hearts,however strongp53 binding tothe BAXpromoter was observed intheMI heart tissue(lane3). MI+SC hearts serve as controls (lane 4). p53 does not bind to BAX promoter in the MI+OxCy cardiac tissue (lane 5). 9 Figure S8. BAX mRNA level in the infarct heart tissue. BAX mRNA levels in MI and treated hearts were measured using RT-PCR. Results show that BAX mRNA is upregulated in the MIhearts. MI hearts treated with mesenchymal stem cell (MI+MSC), oxygenation (MI+OxCy), or combination (MI+MSC+OxCy)abolished BAX mRNA synthesis. 10 FigureS9. BAX protein level in the infarct hearttissue. BAX protein levelsin MI and treated hearts were measured using western blotting. Results show that BAX protein is upregulated in the MI heart. MI hearts treated with mesenchymal stem cell (MI+MSC), oxygenation (MI+OxCy), or combination (MI+MSC+OxCy)abolished BAX protein. 11 FigureS10. In vivo ELISAof p53 modifications. Phosphorylation and acetylation of p53 at the known sites were quantified in the healthy (control), MI and the MI+OxCy hearts.The results show significant increase in p53 phosphorylation of acetylation inthe MI hearts in comparison to the control hearts. Interestinglythe acetylation of p53Lys118 residue is significantlydecreased upon oxygenation of the MI hearts.Datarepresent mean±SD of 7independent measurements. 12 2. SUPPLEMENTALEXPERIMENTALPROCEDURES Western blotting Tissue homogenates were prepared fromthe anterior wall of the leftventricles of rats from all the four groups along with untreated control. After the treatment period, rats were anesthetized and sacrificed at 4 weeks of MI. Control rats (non I/R, n=4) were also used. The hearts were rapidly excised, rinsed in ice-cold PBS at pH7.4 containing 500-U/ml heparin to remove red-blood cells and clots, frozen in liquid nitrogen and stored at -80oC until analysis. The proteins were isolated from tissue samples using Total Protein Extraction kit from Millipore. The tissue homogenate was incubated for 60 min on ice, followed by micro-centrifuging at 10,000× for 15 min at 4oC. Aliquotsof 75 μg of protein from each samplewere boiled inLaemmli buffer (Bio-RadLaboratories, Hercules,CA) containing 1% 2-mercaptoethanol for 5 min. Theprotein was separated by SDS-PAGE, transferred to polyvinylidene difluoride(PVDF) membrane, and probed with primary antibodies for desired protein. The membranes were incubated overnight at 4oCwith the primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Piscataway, NJ) for1h. Themembranes were thendeveloped using DABas substrate(Vector laboratories). Preparationofnuclearand cytoplasmic extracts The tissue was spun at 2000 rpm for 10 min at 4°C and the pallet was collected. This pallet was suspended in 5x volume Buffer A (10 mM HEPES, pH7.9, 4°C, 1 ml; 1.5 mM MgCl2, 150 μl; 10 mMKCl, 500 μl)raise to total volume of 100 ml, 4°C, ice min ~12 ml. Again the pallet was collected by spinning at 2000 RPM 10 min, 4°C. The palette was resuspended in 2x volume Buffer A ~5 ml and again homogenized usingthe homogenizer. The pellet was again collectedby spinning at 2000 RPM 10 min, 4°C. From this pointpellet andsupernatant weretreated separately. A. Pelletconsists of nuclei which weretransferred to ultracentrifugetubes andspun at 16,000 RPM at 4 degrees. The pellet was re-suspend in 0.6 ml buffer C(20 mM HEPES/TRIS 7.9 2 ml, 25%v/v glycerol 50 ml, 0.42 M NaCl 10.5 ml, 1.5 mM MgCl2 150 μl, 0.2 mM EDTA 40 μl, cocktail protease inhibitors 0.5 mM) raise total volume to 100 ml. The pallet was again homogenized and left to stir at 4oC for30 minutes. Again the pellet was collected by spinning at 16000 RPM for 30 minutes. The pellet was incubated with buffer D (20 mM Hepes/Tris 7.9 2ml, 20%v/v glycerol 40 ml, 0.1 M KCl 5 ml, 0.2 mM EDTA 40 μl, protease inhibitor 0.5 mM with total volume raised to 100 ml) for 5 hours. Finally the suspension was spun at 16000 RPM for 20 minutes and the supernatant was collected. The supernatantis thenuclear extract. B. Supernatant is the cytoplasmic extract. To the cytoplasmic fraction 0.11 vol of buffer B ((0.3 M Hepes/tris 7.9 30 ml, 1.4 M KCl 70 ml, 0.03 M MgCl2 3 ml) and total volume raised to 100 ml) was added. The suspensionwas spunat17000RPMfor 4 hoursat4oCand the supernatant was incubated with buffer D for 8 hours. Again the supernatant was collected after spinning the suspension at 17000 RPM for 4hours at 4oC. The supernatantis thecytoplasmic fraction. Immunoprecipitation The proteins wereisolated fromtissue samples using Total ProteinExtraction kitfrom Millipore. After cell lysis the extracted DNA was fragmented by passing the lysed suspension 5to 10 times through a needle attached to a 1-ml syringe. The lysates were pre-cleared to help reduce non-specificbinding of proteins to agarose or sepharose beads.Pre-clearing with an irrelevant antibody or serumwill remove 13 proteins that bind immunoglobulins non-specifically.Thus to the lysate 50 μl of GAPDH (irrelevant) antibody of the same species (rabbit) was added for 3 hours to pre-clear the lysate. To this pre- cleared lysate antibody of NOS3 and p53 were added. Lysate was incubated with these antibodies overnight on ice and then 100 μl of bead slurry was added to the lysate. The bead and lysate mix was incubated for 4 hours at4oCwith gentle agitation. The mixturewas spun at 14,000xg at 4°Cfor10 minutes. The beadpellet was discardedand thesupernatant was keep for immunoprecipitation. In order to increase the yield, the beads were washed 1 or 2 more times in lysis buffer, and the supernatants were collected. True blot anti bodysystem from Affymetrix was used to get rid of heavy and lightchain staining. Annexin-Vstaining Beckton Dickinson flow cytometer was used to detect the apoptotic cell surface shift of phosphatidylserine by the binding of fluorescein isothiocyanate (FITC) conjugated Annexin V to the outer membrane of intact cells. Floating cells were collected by centrifugation and these, as well as the attached cells were washed in PBS (Ca2+ orMg2+ free)/0.1% EDTA. The attached cells were gentlyscraped off the dish and centrifuged. After centrifugation, the cells were treated with 500µl binding buffer (Abcam) then 5 µl of Annexin V-FITC and 5-µlpropidium iodide was added. Cells were treated at room temperature for 5 minutes.These cells were then filtered through 70m mesh, to eliminate cell aggregates and analyzed by flow cytometry. Protein ELISA Sandwich ELISA (in vivo ELISA) from cell or tissue lysate was done as follows; The wells of a PVC microtiter platewere coatedwith100 µl of 5 µg/ml of thedesiredantibody overnight at 4°C.After washing thrice withTBS buffer (0.05%Tween-20in PBS), blocking was done using 5% skimmedmilk in TBS for 2 hours at 4°C. After washing the wells thrice with TBS, 200 µg cell lysate in NP-40 buffer (normal/heat shocked) 11 v/v diluted in 5% skimmed milk in TBS was added to each well and incubatedat 4°Cfor 2 hours. Subsequently100 µl of desiredpolyclonal antibody was added to each well and incubated at 4°C for 2 hours. Again after three quick washes, 100 µl of HRP-conjugated desired secondary antibody was added to each well and kept at RT for another 2 hours. ELISA was developedfor30 minutes andO.D. wasrecordedat 450 nm on Microplatereader (Bio-Rad). Induction of myocardial infarction Male Fisher-344 rats (200-250 g)were used. Rats were randomlydivided into four groups of 8 animals each: (1) MI group (serum-free medium-treated); (2) Ox group (MI with hyperbaric oxygen treatment); (3)MSC (MIwith MSC transplantation); (4)MSC+Ox(MI with MSCand HBO treatment). Rats were anaesthetizedwith ketamine (50 mg/kg, i.p) and xylazine (5 mg/kg, i.p.) and maintained under anesthesia using isofluorane (1.5-2.0%) mixed with air. Myocardialinfarction (MI) was created by ligating the left-anterior-descending (LAD) coronary artery for 1 h. After 1 h of ischemia, the ligation was released,flowwas restored (reperfusion), and thechest cavity was closed. After ligation of the LAD coronary artery, successful infarction was confirmed, for all groups, by an ST elevationon the electrocardiograms. Afterreinstallation of spontaneous respiration, animals were extubated and allowed to recover from anesthesia. No mortality was observed inany of the groups at theend of the study.All theprocedures were performed with the approval of the Institutional Animal Care and Use 14 Committee of The Ohio State University and conformed to the Guide for the Care and Use of Laboratory Animals (NIHPublication No. 86-23). Protocol for oxygen-cycling The rats were subjected to hyperoxygenation treatment by placing them inside acustom-built small- animal hyperbaricchamber (Polyfab; Boston Plastics Manufacturing, Wilmington, MA) connectedto a compressedgas cylindercontaining 100% oxygen. Oxygen (OxCy) was administered daily (100% O2, 2 ATA, 90 min) starting 4 daysafter MI.OxCy was administered 5 daysa weekfor 4 weeks.The animals were placed 1 per cage with up to 3 cages at a time in the chamber. After the chamber was closed, a fill-valve was slowlyopened to allowthe pressure within the chamber to reach 2 ATA (atmospheric pressure absolute) over 5-10 minutes. The valvewas then closed, sealing the chamber and the rats kept in the chamber for 90 minutes under constant observation. After this period, a safety pressure- release valve was manually activated to slowlydepressurize the unit to normal atmosphericpressure over 5-10min. Immunohistochemical staining of cardiac tissue for p53and NOS3 expression Hearts were fixed in formalin and embedded in paraffin. Five-micron sections were cut and preincubated in blocking buffer (5%nonfat dry milkon 0.1 mM PBS)for 45 minutes at 37°C, followed by overnight incubation with the primary antibodies anti-p53 (mouse monoclonal antibody for p53; sc-126; Santa Cruz Biotechnology) oranti-NOS3 (affinity purified rabbit polyclonal antibody for NOS3, sc-654; Santa Cruz Biotechnology) at 4°C. The sections were extensivelyrinsed in 0.05 mM PBS and incubated with peroxidase-conjugated secondary antibodies (1:100) for 2 hours at 25°C. After washing as above, sections were subjectedfor 5 minutestofreshly prepareddiaminobenzidinethat contained H2O2(0.8%).The specimens were gentlyrinsed in 1 PBS, air-dried and mounting medium with DAPI (Vector labs, CA) was applied topically and slides were viewed under fluorescence microscope (Nikon TE2000, Japan).