FIGURE 5.

Annexin V staining demonstrates that lysine 120 acetylation is required for p53-mediated transcription-independent apoptosis. A, H1299 cells expressing either p53ER or the p53ER(K120R) mutant were treated with 100 μg/ml cycloheximide or vehicle for 1 h and subsequently treated with 100 nm 4-OHT or vehicle for an additional 48 h. Following treatment, cells were collected and either subjected to Western blot analysis with the indicated antibodies (left panel) or stained with annexin V-FITC and propidium iodide and then analyzed by fluorescence-activated cell sorter (right panel). Right panel is a graphic representation of annexin V-positive (propidium iodide-negative) cells collected after the treatment indicated. Apoptosis levels were normalized to “fold induction” as discussed in the text, and basal levels of apoptosis are ∼2% in the absence of CHX treatment and 15% in the presence of CHX. Error bars represent standard deviation of three independent reactions. B, H1299 cells expressing either p53 or the K120R mutant under the regulation of a tetracycline-response element were treated with 100 μg/ml α-amanitin or vehicle for 2 h and subsequently administered 500 ng/ml tetracycline or vehicle for an additional 24 h. Following treatment, cells were collected and either subjected to Western blot analysis with the indicated antibodies (left panel) or stained with annexin V-FITC and propidium iodide and then analyzed by fluorescence-activated cell sorter (right panel). Right panel is a graphical representation of annexin V-positive (propidium iodide-negative) cells collected after the treatment indicated. Levels of apoptosis are expressed as fold induction. Basal levels of apoptosis were ∼5% in the absence of tetracycline, which is similar to the level observed in response to α-amanitin treatment of parental H1299 cells. Upon activation of p53 by tetracycline treatment, the number of apoptotic cells reached ∼10% of the total population. Error bars represent standard deviation of three independent reactions.