Pharmacological Modulation of PI3K-Akt-mTOR Pathway

As described previously, flow cytometry was used to examine the phosphorylation of selected mediators in the main track of the PI3K-Akt-mTOR network.^{4 (link)} A detailed methodological description is included in the Supplementary Information. Finally, previous studies have shown that our cryopreserved AML cell populations usually have a viability of 70–75% and a purity of at least 90%;^{15 (link)} this was also seen from the present analyses (live-dead gating; scatter differences).
For pharmacological studies, AML cells were incubated with human insulin 10 µg/mL (Sigma-Aldrich, St. Louis, MO, USA), the PI3K class I inhibitor GDC0941 (Axon Medchem BV, Groningen, The Netherlands), the Akt inhibitor MK2206 (Selleckchem, Houston, TX, USA) or the mTOR inhibitor rapamycin (LC Laboratories, Woburn, MA, USA). AML cells were incubated for 15 minutes with insulin or in medium alone before analysis. For the investigation of pharmacological effects, the cells were incubated with or without insulin for 15 minutes prior to an additional 15-min incubation with a pharmacological agent, followed by flow cytometric analysis. All inhibitors were tested at a final concentration (100 nM) that showed antiproliferative effects in a ³H-thymidine incorporation assay.^{44 (link)}