Manganese superoxide dismutase activity regulates transitions between quiescent and proliferative growth
Article first published online: 10 MAR 2008
© 2008 The Authors. Journal compilation © Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2008
Volume 7, Issue 3, pages 405–417, June 2008
How to Cite
Sarsour, E. H., Venkataraman, S., Kalen, A. L., Oberley, L. W. and Goswami, P. C. (2008), Manganese superoxide dismutase activity regulates transitions between quiescent and proliferative growth. Aging Cell, 7: 405–417. doi: 10.1111/j.1474-9726.2008.00384.x
- Issue published online: 10 MAR 2008
- Article first published online: 10 MAR 2008
- Accepted for publication 20 February 2008
Fig. S1 Appropriate manganese superoxide dismutase (MnSOD) activity is required for quiescent fibroblasts’ re-entry into the proliferative cycle. Quiescent normal skin human fibroblasts (NHFs) were mock-infected or infected with 30 MOI of adenoviruses containing wild-type or dominant negative mutant form of human MnSOD cDNA. Equal amounts of total protein extracts were analyzed for MnSOD activity using native gel electrophoresis (inset). Cells from replicate dishes were replated at a lower density and continued in culture for 6 days; cell numbers counted at indicated times. Cell numbers represent total number of cells per 60-mm dish. Asterisk indicates significant difference in cells infected with adenovirus containing human mutant MnSOD cDNA (AdmMnSOD) compared to control and AdMnSOD infected cells at 4 and 6 days postplating (n = 3, p < 0.05).
Fig. S2 Specificity of the DCFH (5-(and 6)-chloromethyl-2′,7′-dichlorodihydro fluorescein diacetate) assay for measurements of intracellular hydrogen peroxide steady-state levels. (A) Representative histograms of DCFH mean fluorescence intensity (MFI): asynchronous cultures of manganese superoxide dismutase (MnSOD) (+/+) mouse embryonic fibroblasts (MEF) were mock infected or infected with adenoviruses containing 30 MOI of human MnSOD (AdMnSOD), catalase (AdCAT), and glutathione peroxidase 1 (AdGPx1) cDNAs. Infected cells were washed with Hanks’ buffer salt solution (HBSS) and incubated with 1 µg mL−1 DCFH (Invitrogen) for 15 min. Monolayers were trypsinized and resuspended in HBSS containing 10% fetal bovine serum (FBS). DCFH fluorescence was measured by flow cytometry. (B) Mean fluorescence intensity was analyzed using Flowjo software and fold-change calculated relative to mock-infected control. Auto-fluorescence of unlabeled cells was used for background fluorescence correction. Inset shows immunoblotting of MnSOD, CAT and GPx1 in mock-infected control and adenovirus-infected cells. Asterisk indicates significant difference in AdMnSOD-infected cells compared to control and AdBgl II-infected cells (n = 3, p < 0.05).
Fig. S3 Specificity of the electron paramagnetic resonance (EPR) assay for measurements of superoxide steady-state levels. Asynchronous cultures of manganese superoxide dismutase (MnSOD) (+/–) MEFs were mock infected or infected with 100 MOI of adenoviruses containing an empty vector (AdBgl II) or MnSOD cDNA (AdMnSOD). Cultures at 72 h postinfection were rinsed with phosphate-buffered saline (PBS) and incubated with buffer containing 100 mM 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Monolayer cultures were scrape-harvested and transferred to flat cell for EPR spectroscopy. EPR peak height calculated and normalized to 1 million cells. Asterisk indicates significant difference in AdMnSOD-infected cells compared to mock and AdBgl II-infected cells (n = 3, p < 0.05).
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