Gdc 0941

LY294002 was from Sigma-Aldrich (St. Louis, MO, USA), GDC-0941 and CT98014 were from Axon Medchem (Groningen, Netherlands), Annexin-V-FITC was generated in our lab. Annexin-V-APC (RUO) was from Becton Dickinson (Franklin Lakes, NJ, USA). 4-OHT was from Sigma-Aldrich (St. Louis, MO, USA).
IL-2 and IL-3 were from Peprotech (Rocky Hill, NJ, USA).
The following antibodies were used for western blotting: Puma (#3043) was from Prosci (Poway, CA, USA), human Puma (#12450), Foxo3a (#2497) were from Cell Signaling Technologies (Danvers, MA, USA). GSK3a/b (sc-56913), 14-3-3 (sc-1657) and NFATc1 (sc-7294) were from Santa Cruz (Dallas, TX, USA). Tubulin (MCA77G) was from Bio-Rad (Hercules, CA, USA). MCL1 (600-401-394S) was from Rockland (Limerick, PA, USA).
FLAG-M2 agarose affinity beads were from Sigma-Aldrich (St. Louis, MO, USA).

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)}

The class I PI3K inhibitor, GDC-0941 (Axon Medchem), was used to treat bladder cancer cell lines and TERT-NHUC. The dose range chosen was based on previous studies that report IC₅₀ values of 0.28–0.95 μM for cell viability of solid tumor cell lines, as well as pharmacokinetic data available from phase I clinical studies that report a maximum of 2 μM GDC-0941 plasma concentration in patients [30 (link), 41 (link)]. Cell viability was assessed by CellTiter-Blue® (Promega) analysis of bladder cancer cell lines and TERT-NHUC subjected to GDC-concentrations from 0 to 2 μM. Cell viability was assessed by CellTiter-Blue® (Promega) analysis. 1000–4000 cells per well (number of cells determined to ensure that confluence was not achieved in untreated controls during the experiment) were plated in 96-well plates in five replicate wells and allowed to attach for 24 h before addition of 0–2 μM GDC-0941 in 0.1 % DMSO. After 72 h, 20 μl of CellTiter-Blue solution was added to the medium for 2 h and fluorescence read at 550 nm. Medium alone was used as a blank. Prism software (GraphPad Software, La Jolla, CA, USE) was used to calculate IC₅₀ values.
Cell cycle and apoptosis analysis of cells cultured with 1 μM GDC-0941 or DMSO only for 48 h was evaluated by flow cytometry as described [40 (link)]. All assays were done in triplicate and repeated at least three times.

The MEK inhibitor PD184352 (in short PD) and GDC-0941 were purchased from Axon Medchem and Selleckchem, respectively. The PI3K inhibitor LY294002, the EGFR inhibitor AG1478 and cisplatin were obtained from Calbiochem. ON-TARGET plus SMART pool siRNA for BBC3 (PUMA), SPRED1, SPRED2 and non-targeting siRNA were from Thermo Scientific. For transfection of siRNA, X-tremeGENE siRNA Transfection Reagent (Roche) was used according to the manufacturer's instructions.

Reagents were from Sigma (St. Louis, MO) unless noted. Iodoacetate, lactate, pyruvate, and cycloheximide stocks were prepared in water. Rotenone, oligomycin A, and AICAR were dissolved in DMSO. BEZ235 (Axon Medchem, Reston, VA), Torin-1 (Tocris Bioscience, Bristol, UK), Torin-2 (Selleck, Houston, TX), BKM120 (Axon Medchem), GDC0941 (Axon Medchem), Gefitinib (Axon Medchem), MK2206 (Selleck), PD 0325901 (Sigma and Selleck), and Rad001 (SU2C PI3K Dream Team Mouse Pharmacy, which obtains compounds from Shanghai Haoyuan Chemexpress; [Elkabets et al., 2013 (link)]) were dissolved in DMSO. For GF titration, EGF (Peprotech, Rocky Hill, NJ) and insulin (Sigma) were diluted in PBS and added at indicated concentrations.