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Figure 3


Fig. 3. R246S mutant protein does not affect differentiation of ES cells but also exerts DN effects in the differentiated state. (A) Pluripotency of ES cells is not affected by expression of R246S mutant p53. The expression of various stem-cell markers was analyzed by semi-quantitative RT-PCR (left panel). The morphology of the undifferentiated ES-cell colonies was routinely monitored by phase-contrast microscopy and representative images of ES cells of various p53 genotypes are shown (right panel). (B) The expression of stem-cell markers was analyzed during retinoic-acid-induced (0.1 µM) differentiation of ES cells. (C) Localization of p53 in ES cells differentiated with 0.1 µM retinoic acid for 6 days, after treatment with (+{gamma}) or without (–{gamma}) 20 Gy irradiation, was analyzed by fluorescence confocal microscopy. The green fluorescence represents p53 protein; red fluorescence represents genomic DNA. (D,E) Differentiated ES cells were irradiated with 20 Gy and harvested 24 hours later. (D) Cell-cycle analysis was performed by flow cytometry and ModFit cell cycle analysis software. (E) Cellular proliferation was determined by BrdU staining of cells (treated without or with doxorubicin) followed by flow-cytometric analysis. Data represents the percentage reduction of S-phase cells (D) or the percentage of BrdU+ cells (E) (mean ± s.e.m.) from three independent experiments, each performed in duplicate.





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