Bms 754807

Human dermal microvascular endothelial cells (HMEC-1) and human embryonic kidney cells 293 (HEK293) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HMEC-1 were cultured in MCDB131 medium (Gibco, Thermo Fisher Scientific Inc., Waltham, MA) supplemented with 10% fetal bovine serum (FBS, Gibco), 2 mM l-glutamine (Gibco), 10 ng/mL epidermal growth factor (EGF, Sigma-Aldrich, St. Louis, MO) and 1 μg/mL hydrocortisone (Sigma-Aldrich). HEK293 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco) supplemented with 10% FBS (Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin sulfate (Gibco). Both cell types were cultured in a humidified atmosphere (5% CO₂, 37 °C), with medium changes twice a week. BMS-754807 (referred to as RI in this article), a selective small molecule receptor inhibitor of IGF-1R and InsR (Carboni et al., 2009 (link); Wittman et al., 2009 (link); Kolb et al., 2011 (link)), was obtained from Selleck (Houston, TX) and used at a concentration of 1 μM for in vitro assays.

Human cell lines included an NF2-null immortalized MN line Ben-Men-1, NF2 AC-CRISPR, and two independent human primary MN for which cell line establishment and growth conditions have been previously described (15 (link), 54 (link)). All primary cultures were collected following Massachusetts General Hospital Human Subjects protocols for tumor acquisition after informed consent. In addition, we generated an immortalized human MN line, MN1-LF from an independent NF2-null human primary MN cell line derived from a surgical resection. Immortalization methods and confirmation was carried out on a fee-for-service basis by Alstem, Inc. Briefly, 1 × 10⁵ cells were transduced with lentivirus encoding SV40 large T antigen and a puromycin resistance gene. Cells were infected at a multiplicity of infection of 2. Following selection with 1.5 μg/ml puromycin for 3 days, cells underwent an additional three passages. PCR amplification of SV40 was performed to confirm immortalization followed by expansion and freezing of cells.
Reagents included exogenous heregulin/NRG1 (Sigma); inhibitor drugs lapatinib, erlotinib, INK128/TAK-228, and BMS-754807 (Selleck Chemicals); rapamycin (EMD Millipore); AZD2014 (obtained from AstraZeneca); and MM-121 (generously provided by Merrimack Pharmaceuticals). Drug treatment concentrations and times are described in the figure legends.

Linstinib (OSI-906), PF-562271, and BMS-754807 were purchased from Selleckchem. Cells were seeded at 40% confluency overnight and incubated with drugs dissolved in 0.1% DMSO for 48 h. Viability was measured by the CellTiter 96 AQueous One Solution cell proliferation assay (Promega, Southampton, UK) following manufacturer’s instructions. The optical absorbance was recorded at 490 nm using a BioTek 800 TS microplate reader (Agilent, Cheadle, UK). Data were normalised against the vehicle control.