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Fig. 4. Conditional activation of p53 causes a reduction in AKT membrane localization. (A) OSEC2 cells at 34°C in the absence of serum stimulation did not show membrane localization of AKT-PH-GFP protein. Upon serum stimulation (10% FBS) at 34°C, the AKT-PH-GFP protein was targeted to the membrane, indicative of increased PI3K activity in the absence of functional p53. When serum-stimulated OSEC2 cells at 34°C were treated with 20 µM of the PI3K inhibitor LY294002 for 1 hour, membrane localization was minimal. OSEC2 cells with functional p53 at 39°C did not demonstrate membrane localization in either the absence or presence of serum stimulation. Similar to the OSEC2 cells, IOSE 166h (B) and IOSE 166a (C) cells showed a significant reduction in AKT-PH-GFP membrane localization at 39°C compared with at 34°C after serum stimulation. (D) Between 300 and 400 cells per group in each cell line (OSEC2, IOSE 166h and IOSE 166a) in the preparations shown in A, B and C were grouped by percentage into three categories: strong, weak and no membrane localization (ML). Only with serum stimulation at 34°C was there a highly significant increase in the percentage of cells with strong membrane localization (P<0.001). Statistical analysis was by one-way ANOVA test (Tukey).
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