Figure 3.

Pharmacodynamic responses to PI3K isoform selective inhibition. The relative levels of phosphorylated AKT in NSCLC cell lines (A549, H460, H1975, PC9, H1650) treated with increasing concentrations of PI3K inhibitors (A) A66 (B) TGX-221 (C) IC488743 and (D) CAL-101. Cells were synchronized by serum-starved for 24hr prior to release in RPMI 1640 media containing 1% serum with or without drug for 3 hr. Cell lysates were collected for immunoblotting analysis with pAKT (S473) and tAKT antibodies. α-tubulin serves as the normalization control. ImageJ software was used for densitometric analysis of pAKT and tAKT levels. Three independent experiments were quantified, pAKT (S473) was normalized to tAKT , and data were averaged to reflect relative changes in pAKT (S473) activation. Error bars represent mean ± SEM. PIK3CA mutant cell lines are indicated.