Figure 6

PTEN expression in cells with tetracycline-regulated p53 MDAH cells (which are p53-negative), or the same cells harboring a tetracycline-regulated p53 gene (TR9-7), were kept in monolayer culture in the absence or presence of tetracycline (1 μg/ml) for 24 hours. TR9-7 cells contain the tet-off system, i.e., in the presence of tetracycline, p53 is off (not expressed), whereas in the absence of tetracycline, p53 is expressed [42]. After 24 hours of incubation with or without tetracycline, the cells were transferred to suspension culture in the continued presence or absence of tetracycline. At 0, 12 and 24 hours after the onset of suspension culture, cellular lysates were prepared and analyzed by Western blot for the expression levels of PTEN as indicated. In order to verify proper control of p53 by tetracycline, the amount of p53 protein in each lysate was analyzed as well (shown in the bottom panel). Furthermore, to establish that the elevated levels of p53 were indeed functional, the increased expression of one of its target genes, p21^Cip1[65], was confirmed by Western blot analysis; in this case, p21^Cip1 was found to be highly expressed in TR9-7 cells in the absence of tetracycline, but not in TR9-7 cells in the presence of tetracycline, nor in MDAH cells with or without tetracycline (not shown).