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Embryonic Stem Cells/Induced Pluripotent Stem Cells
Article first published online: 24 JUL 2012
Copyright © 2012 AlphaMed Press
Volume 30, Issue 8, pages 1634–1644, August 2012
How to Cite
Hishida, T., Nozaki, Y., Nakachi, Y., Mizuno, Y., Iseki, H., Katano, M., Kamon, M., Hirasaki, M., Nishimoto, M., Okazaki, Y. and Okuda, A. (2012), Sirt1, p53, and p38MAPK Are Crucial Regulators of Detrimental Phenotypes of Embryonic Stem Cells with Max Expression Ablation. STEM CELLS, 30: 1634–1644. doi: 10.1002/stem.1147
Author contributions: T.H.: conception and design, data analysis and interpretation, and manuscript writing; Y. Nozaki, Y. Nakachi, Y.M., H.I., M. Katano, M. Kamon, M.H., M.N., and Y.O.: data analysis and interpretation; A.O.: conception and design, financial support, and manuscript writing.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS June 13, 2012.
- Issue published online: 24 JUL 2012
- Article first published online: 24 JUL 2012
- Accepted manuscript online: 13 JUN 2012 02:37PM EST
- Manuscript Accepted: 31 MAY 2012
- Manuscript Received: 29 FEB 2012
- Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan
- Strategic Research Center in Private Universities to the Saitama Medical University Research Center for Genomic Medicine
Additional Supporting Information may be found in the online version of this article.
|SC_12-0205_sm_supplFigure1.tif||959K||Supporting information Figure 1. SB239063, a structural analog of SB203580, also showed a prominent activity to mitigate the detrimental phenotype of Max-null ESCs. SB239063 is a more selective inhibitor of p38MAPK than SB203580 and was used to examine its effect on alleviating the cell death of Max-null ESCs as shown in Figure 1B at the indicated concentrations. After culturing for 12 days, cells were inspected microscopically (upper panel) and then subjected to Leishman staining (lower panel).|
|SC_12-0205_sm_supplFigure2.tif||687K||Supporting information Figure 2. Evaluation of the abilities of PFTα, Nam and SB239063 to preserve Oct3/4 expression after ablation of Max expression in ESCs. Max-null ESCs treated as indicated were subjected to flow cytometric analyses to detect Oct3/4 after permeabilization with 90% methanol as described in Figure 4A.|
|SC_12-0205_sm_supplFigure3.tif||902K||Supporting information Figure 3. Effect of Nam, Sirtinol, DPQ and PD0325901 on expression levels of pluripotency marker genes in Dox-untreated control Max-null ESCs. (A): Estimation of Oct3/4 protein levels by flow cytometry. Analyses were performed as described in Figure 4A. (B): Western blot analyses to evaluate the effects of chemicals on the levels of Oct3/4, Sox2 and Nanog in Dox-untreated Max-null ESCs.|
|SC_12-0205_sm_supplFigure4.tif||566K||Supporting information Figure 4. Nam-dependent alleviation of changes of gene expression in ESCs coupled to ablation of Max expression. (A): Comparison of the effect of Max expression ablation on the global gene expression profile between Nam-treated and -untreated ESCs. Left and right panels show the scatter plot of Dox-treated (6 days) and -untreated Max-null ESCs that were untreated and treated with Nam, respectively. Blue and gray spots indicate the expression signal change is more than 2-fold and less than 2-fold compared between sample pairs, respectively. The variance was calculated and is shown in the lower right of each panel. (B): Effect of Nam on preventing up-regulation of differentiation marker genes in ESCs coupled to ablation of Max expression. RNA was prepared from Max-null ESCs under the indicated conditions and used for quantitative RT-PCR.|
|SC_12-0205_sm_supplFigure5.tif||163K||Supporting information Figure 5. Only marginal elevation of p53 activity was evident in Dox-treated Max-null ESCs. The luciferase reporter gene bearing the cyclin G1 promoter that carries two p53 binding sites was introduced together with the internal control pRL/TK vector into Max-null ESCs pretreated or untreated with Dox for 2 days. Culture conditions were maintained after plasmid introduction for 24 hrs. Then, cell extracts were prepared and dual luciferase reporter assays were performed. Data are the mean with standard deviation (n=3). Activities obtained from Dox-untreated cells were arbitrarily set to one.|
|SC_12-0205_sm_supplFigure6.tif||505K||Supporting information Figure 6. Effect of Sirt1 and p53 knockdown on the protein levels of Max-interacting Myc family proteins and the transcriptional repressor Mad3. Western blot analyses of extracts from Max-null ESCs under the indicated conditions were performed as described in Figure 6C.|
|SC_12-0205_sm_supplTable1.tif||51K||Supplementary Table 1|
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