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Figure 2


Fig. 2. Conditional activation of p53 causes a decrease in p110{alpha} protein and PIK3CA transcript levels. (A) Western blot analyses of ts OSEC2, IOSE 166h and IOSE 166a cells show that, after a switch from 34 to 39°C, p53 became functional, p21 and PTEN levels increased, whereas p110{alpha} levels decreased. IOSE lines with non-functional p53 (IOSE 80pc and IOSE 397) and WI38 cells with wild-type p53 were used as controls to rule out direct effects of temperature on p110{alpha} levels. There was no decrease in p110{alpha} levels with the increase in temperature in these non-ts control cells. WI38 cells expressed low levels of p110{alpha} protein; these levels did not change in response to increased temperature. Furthermore, in the control cells, PTEN levels remained unchanged (IOSE 80pc) or decreased (IOSE 397 and WI38) at 39°C. +/–indicate functional/non-functional SV40 TAg or p53. (B) Real-time quantitative RT-PCR of the cells shown in A demonstrates that, upon temperature shift to 39°C, which results in p53 activation, PIK3CA transcript levels significantly decreased in the ts lines (OSEC2, IOSE 166h, IOSE 166a). PIK3CA transcript levels increased at 39°C in non-ts control cells (IOSE 80pc, IOSE 397 and WI38). At 34°C, at which p53 remained inactive, all the ts cell lines had higher PIK3CA transcript levels on day 5 at 34°C compared to day 1 at 34°C. The expression level of the ribosomal RNA gene was used as control.





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