Fig. 3

Differential Akt/mTOR pathway suppression in TfRCC cells treated with dual mTORC1/mTORC2 versus selective mTORC1 inhibition. A representative Western blot shows time- and dose-dependent effects of dual mTORC1/2 inhibition with AZD8055 versus selective mTORC1 inhibition with sirolimus in a TfRCC cell line (UOK146). Cells were cultured with 0–500 nM of either drug for 0, 1 and 6 h. Dose- and time-dependent reductions by AZD8055 treatment in levels of phosphorylated S6 or 4EBP1 and Akt (Ser473) confirmed target inhibition of mTORC1 and mTORC2, respectively, with complete suppression of each achieved with 500 nM by 6 h. Similar dose- and time-dependent suppression was observed for other Akt/mTORC pathway members, including phosphorylated GSK3β, phosphorylated mTOR and HIF1α. In contrast, sirolimus achieved complete suppression of phosphorylated S6 by 6 h, but caused time- and dose-dependent increases in other Akt/mTOR pathway members consistent with feedback activation