Nci h727

Pancreatic neuroendocrine tumor cell lines BON-1 [39 (link)] and QGP-1 [40 (link)] (both obtained from Japanese Collection of Research Bioresources) and the typical bronchial carcinoid-NET cell line NCI-H727 (H727) [41 (link)] (purchased from ATCC, Manassas, VA) underwent authentication at the DSMZ (Braunschweig, Germany, in 2014), and their neuroendocrine features were confirmed by immunocytochemistry. Participating researchers received cell aliquots of the same passage together with SOPs for culture procedures. Cell lines had been tested for PIK3CA mutations by panel sequencing (Ion Seq Torrent Lung and Colon Panel v2). No PIK3CA mutations had been detected. BYL719 is a selective PI3Kα inhibitor developed by Novartis Pharma. All cell lines have been treated with different concentrations (10 nM– 250 μM) of BYL719 (kindly provided by Novartis) versus DMSO control for several time periods according to assay type. Everolimus was purchased from Selleckchem (Munich, Germany) and used in concentrations between 1 and 10nM.

Human pancreatic NET cell line BON1 [9 (link)] (kindly provided by R. Göke, University of Marburg, Marburg, Germany) and pancreatic islet tumor cell line QGP-1 [9 (link)] (originally obtained from the Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan), were both maintained in Dulbecco’s modified Eagle’s medium/F12 (at a ratio of 1:1) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 0.4% amphotericin B in a 37 °C humidified incubator with 5% CO₂. Human bronchopulmonary neuroendocrine NCI-H727 tumor cells [9 (link)] (originally obtained from ATCC, Manassas, VA, USA) and the human small intestinal NET cell line GOT1 [9 (link),39 (link)] (kindly provided by O. Nilsson, Sahlgrenska University Hospital, Göteborg, Sweden) were both cultured in Roswell Park Memorial Institute medium-1640 (RPMI-1640) supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.4% amphotericin B in a 37 °C humidified incubator with 5% CO₂.
To treat NET cells with WNT974 (also named LGK974; Novartis, Basel, Switzerland) or PRI-724 (Selleckchem, Germany), the cell lines were first seeded into cell culture dishes and grown overnight prior to treatment with various doses of WNT974 (1–32 µM, dissolved in dimethyl sulfoxide (DMSO)) or PRI-724 (1–10 µM, dissolved in DMSO) for different periods of time according to the assays listed below.

T24 (bladder carcinoma), SKOV3 (ovarian adenocarcinoma), A549 (lung carcinoma), NCI‐HT29 (colorectal adenocarcinoma) and NCI‐H727 (lung carcinoid) were all purchased from ATCC (Manassas, VA). Mel526 (melanoma cell line) was a kind gift from Dr Magnus Essand and PEA2 (ovarian adenocarcinoma) was a kind gift of Dr Yumeng Mao, both from Uppsala University, Sweden. MiaPaca2 (pancreatic cell line) was a kind gift from Dr Rainer Heuchel (Karolinska Institute, Sweden). T24 and MiaPaCa2 were cultured in Dulbecco's Modified Eagle Medium (DMEM), NCI‐H727, A549, PEA2 and Mel526 were cultured in Roswell Park Memorial Institute (RPMI)‐1640 medium and NCI‐HT29 and SKOV3, were cultured in McCoy's 5A medium. All culture media were supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/mL)/streptomycin (100 μg/mL) (PeSt). For A549 and PEA2, 1% of sodium pyruvate was added to the cultures. All reagents were purchased form Invitrogen, Carlsbad, CA, USA.