Zstk474

Cells were seeded in 96-well plates at 5000 cells per well 24 hours prior to treatment. Cell viability was assessed 48 and 72 hours after treatment using CellTiter-Glo (Promega). Luminescence was normalized to vehicle treatment. All cell viability assays were done in triplicate for each experiment and repeated at least three times. GI₅₀ values, defined as treatment dose at which cell viability was decreased to 50% of vehicle treatment condition, were estimated from log (agonist) versus response including variable slope (four parameters) statistics in GraphPad Prism. Separate cell viability assays were performed to analyze whether any of four novel PI3K agents—ZSTK474 (LC Laboratories), BKM120, GDC-0032, and BYL719 (provided by the Developmental Therapeutics Program at the National Cancer Institute)—demonstrated more potent inhibitory effects on DIPG. In addition, assays were used to determine whether combination therapy with ZSTK474 and trametinib (Developmental Therapeutics Program at the National Cancer Institute) would result in synergistic inhibition of DIPG. Each cell line was treated with each drug either individually or in combination. Combination index (CI) was calculated using Compusyn software (Combosyn) using the Chou-Talalay method.^{22 (link)}

AT9283, AZD4547, AZD6244, BGJ398, crizotinib, pictilisib, ibrutinib, PD173074, ruxolitinib, TAE684, and gandotinib were all purchased from Selleck Chemicals, AUY922, dovitinib, everolimus, gefitinib, mubritinib, saracatinib, sunitinib, and ZSTK474 were from LC Laboratories, CH5424802 was from Active Biochem, SB525334 was from Tocris, and fedratinib was from Axon Medchem. The human gastric cancer cell line SNU-16 was obtained from ATCC and was cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum according to the manufacturer’s instructions.

To mimic loss of attachment, cells were seeded in culture dishes coated with polyHEMA (Sigma-Aldrich) to prevent cell adherence for various period of time. The cell suspensions were placed into regular culture dishes 16 h prior to end-point viable cell counting. To identify the signaling pathways that promote survival of detached cells, a panel of chemical inhibitors were added in culture medium: PTK2/FAK inhibitor – 0.5 μM PF562271 (Selleckchem, Houston, TX, USA), ERK1/2 inhibitor – 10 μM U0126 (LC Laboratories, Woburn, MA, USA) or 10 μM PD98059 (Santa Cruz Biotechnology, Dallas, TX, USA), AKT inhibitor – 30 μM ZSTK474 (LC Laboratories), TGFBRI/II inhibitor – 10 μM LY210761 (Selleckchem), SRC inhibitor – 3 μM dasanitib (LC Laboratories) or Notch inhibitor – 5 μM DAPT (Selleckchem).