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Additional Supporting Information may be found in the online version of this article.

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SC_12-0205_sm_supplFigure1.tif959KSupporting 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.tif687KSupporting 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.tif902KSupporting 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.tif566KSupporting 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.tif163KSupporting 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.tif505KSupporting 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.tif51KSupplementary Table 1

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