Fig. 2. Conditional activation of p53 causes a decrease in p110 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 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 levels. There was no decrease in p110 levels with the increase in temperature in these non-ts control cells. WI38 cells expressed low levels of p110 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.