SEARCH

SEARCH BY CITATION

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

FilenameFormatSizeDescription
emmm201202055.reviewer_comments.pdfPDF document787KReview Process File
emmm201202055-sm-0002-SuppFig-S1.pdfPDF document658K

Figure S1. Purity check on the nuclear 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 lack of tubulin protein in the nuclear fraction and lack of PARP in the cytoplasmic fraction.

Figure S2. Diagrammatic representation of pNOS3p-luc1 vector construct. The 602 base-pair NOS3 promoter, upstream of the transcription start site is shown.

Figure S3. 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 increase in pNOS3p-luc1 in resveratrol-treated H4TG cells. p53 gene-silencing using p53 siRNA abolished the activation of p53 promoter. Data represent mean ± SD of eight independent measurements. *p = 2.1E−09.

Figure S4. Role of p53 in the upregulation of NOS3 gene in 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.

Figure S5. Diagrammatic representation of the minimal pNOS3p-luc2 vector construct. pNOS3p-luc2 (NOS3-p53RE) and the mmpNOS3p-luc2 (mutant minimal promoter2, with mutated p53RE sequence) vector constructs are shown.

Figure S6. Co-localization of p53 and NOS3 proteins in the infarct 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.

Figure S7. Binding of p53 protein on the BAX gene promoter. Chromatin immunoprecipitation (ChIP) was used to analyse the binding 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 strong p53 binding to the BAX promoter was observed in the MI heart tissue (lane 3). MI + SC hearts serve as controls (lane 4). p53 does not bind to BAX promoter in the MI + OxCy cardiac tissue (lane 5).

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 MI hearts. MI hearts treated with mesenchymal stem cell (MI + MSC), oxygenation (MI + OxCy) or combination (MI + MSC + OxCy) abolished BAX mRNA synthesis.

Figure S9. BAX protein level in the infarct heart tissue. BAX protein levels in 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.

Figure S10. In vivo ELISA of 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 in the MI hearts in comparison to the control hearts. Interestingly the acetylation of p53Lys118 residue is significantly decreased upon oxygenation of the MI hearts. Data represent mean ± SD of seven independent measurements.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.