Pik 90

Kelly, SH-SY5Y, SHEP, SK-N-SH, IMR32, SKN-Be2C, IMR5 and SK-N-AS human neuroblastoma cell lines were obtained from the University of California at San Francisco Cell Culture Facility (San Francisco, CA) and from the American Type Culture Collection (Manassas, VA). Cells were grown in DMEM or RPMI containing 10% fetal bovine serum (PAA “Gold”). In specified experiments, cells were serum starved in 0.2% FCS for 6 h before analysis and treated with recombinant human insulin-like growth factor-1 (IGF-1; Sigma) at 50ng/mL for 30 min before harvesting. NVP-BEZ235 (Novartis), Torin1 (Nathaniel Gray), staurosporine (Alexis Biochemicals), ZSTK474 (Alexis Biochemicals), GSK3β inhibitor (Calbiochem), TGX221 (Selleck chemicals), PIK90 (Selleck chemicals) and Rapamycin (Selleck chemicals) were all prepared as a 10mM stock solution in 100% DMSO. Working solutions were prepared freshly by dilution in 100% DMSO prior addition to the cell media at a final concentration of 0.1% DMSO. SHEP cells were stably transfected with constructs wild-type or mutant for MYCN and appropriate clones were screened and selected essentially as described previously [37 (link)]. Cells were regularly screened for Mycoplasma using a PCR-based assay (VenorGem, Minerva Biolabs).

Human T-ALL cell lines Jurkat and Loucy (PTEN deleted), DND-41 and ALL-SIL (PTEN non deleted) were cultured in RPMI-1640 medium (Life Technologies Italia, Monza, Italy) supplemented with 10–20% fetal bovine serum (Life Technologies), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, Saint Louis, MO, USA) at 37°C in a humidified atmosphere of 5% CO₂. PI3K inhibitors BKM-120, PIK-90, ZSTK-474, A-66, TGX-221, AS-605240, CAL-101 and IPI-145, the caspase inhibitor z-VAD and the autophagy inhibitor 3-MA, were from Selleck Chemicals (Houston TX, USA).

OVCAR8, OVCAR4, OVCAR3, EFO21, and DOV13 were obtained from National Cancer Institute. SKOV3 was from American Type Culture Collection. The cell lines were maintained in RPMI-1640 media (Gibco, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS) (Gibco), and 1% penicillin–streptomycin at 37 °C and 5% CO₂. Cell lines were authenticated using short tandem repeat DNA profiling and tested with negative mycoplasma contamination. PI3K inhibitors (GDC-0941, PIK-90, A66, BYL719, and TGX-221), AKT inhibitors (MK-2206 and GDC-0068), and AXL inhibitor (BGB324) were obtained from Selleckchem (Houston, TX). AXL inhibitor (TP-0903) was purchased from ApexBio (Houston, TX). PDK1 inhibitors (OSU-03012 and GSK2334470) and SGK1/2 inhibitor (GSK650394) were purchased from Santa Cruz Biotechnology (Dallas, TX). The used doses of the inhibitors in vitro are as follows: GDC-0941, 10 µM; PIK-90, 10 µM; A66, 2 µM; BYL719, 2 µM; TGX-221, 10 µM; MK-2206, 5 µM; GDC-0068, 5 µM; BGB324, 1 µM; TP-0903, 0.5 µM; OSU-032012, 5 µM; GSK2334470, 2 µM; and GSK650394, 10 µM.