Qgp 1

The human neuroendocrine cell lines BON and QGP-1 (pancreatic), LCC-18 (colonic), and H727 and UMC-11 (pulmonary) were cultured in RPMI 1640 containing 10% fetal calf serum in a humidified atmosphere at 37°C with 5% CO2. BON cells were a kind gift of Courtney Townsend (University of Texas, Galveston, TX, USA). LCC-18 cells were kindly donated by Kjell Öberg (University of Uppsala, Sweden). QGP-1 were obtained from the Japanese Collection of Research Bioresources, and H727 and UMC-11 from ATCC (Manassas, VA, USA). Cells were cultured for no longer than 15 passages. All human cell lines have been authenticated by DSMZ (Braunschweig, Germany) using STR profiling.

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.

QGP-1, a human pNET cell line, was purchased from the Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan). NIT-1, a mouse pNET cell line, was purchased from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). The passages of the two cell lines used in this study were less than 15. We sent the QGP-1 cell line to the Center for Genomic Medicine of National Cheng Kung University for genotyping in June 2016, and the result showed the same STR PCR DNA profile as those in the JCRB database. QGP-1 and NIT-1 cells were cultured in RPMI-1640 (HyClone, South Logan, Utah, USA) medium and F12-Kaighn's (Gibco, Grand Island, NY, USA) medium, respectively, containing 10% fetal calf serum and antibiotics. PTEN and c-Myc expression plasmids were purchased from Addgene (Cambridge, MA, USA). shRNAs targeting PTEN, LKB1, AKT and c-Myc were obtained from the National RNAi Core Facility of Academic Sinica (Taipei, Taiwan). Rapamycin and RAD001 were purchased from LC Laboratories (Boston, MA, USA) and Selleckchem (Houston, TX, USA), respectively. Metformin was purchased from TOCRIS (Bristol, UK), and 10058-F4 was purchased from Calbiochem (San Diego, CA, USA).

HEK (human embryonic kidney) 293 cell and QGP-1 (a human cell line derived from pancreatic islet cell carcinoma) were supplied from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). HEK293 and QGP-1 cells were maintained at 37°C, in 5% CO₂ with high glucose DMEM (HEK293) or RPMI-1640 (QGP-1) (Wako, Osaka, Japan) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 100 units/ml penicillin (Wako), and 0.1 mg/ml streptomycin (Meiji Seika, Tokyo, Japan).

The following GEP-NEN cell lines were used for in vitro experiments: pancreatic: BON [58 (link)] and QGP-1 [59 (link)]; obtained from Japanese Collection of Research Bioresources), ileal: KRJ-1 [60 (link)] and colonic: LCC-18 [61 ].
All cell lines were authenticated (if indicated as unique) by genetic STR typing by the DSMZ, Braunschweig, Germany in 2012 and 2013 and only cells passaged not longer than 15 passages after receipt were used. Cells were tested periodically for maintained cell line specific expression of neuroendocrine markers (chromogranin A, synaptophysin, cytokeratin, vimentin, syntaxin) by immunofluorescence microscopy. All cell lines were grown in Quantum 263 for tumor cells (GE Healthcare Munich, Germany) including 1% penicillin/streptomycin or in cell line specific medium (containing 10% FBS gold and 1% penicillin/streptomycin): BON and KRJ-1 in Dulbecco's Modified Eagle Medium: Nutrient Mixture 1:1 F-12 (DMEM/F12) with stable glutamine, QGP-1 were cultured in RPMI 1640 with stable glutamine and LCC-18 were grown in DMEM 4.5g/l glucose with stable glutamine. The non-neuroendocrine HT-29 and the FOXM1 overexpressing U2OS cell lines were used as controls, if indicated.

Human pancreatic NET cell lines BON (BON, provided by Dr. Mark Hellmich, The University of Texas Medical Branch at Galveston) and QGP-1 (obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRBC)) were cultured, according to our routine and standard protocol. Briefly, BON or QGP-1 cells were, respectively, grown in glutamine-containing DMEM: F-12 media (Gibco) or RPMI1640 (Corning, Lawrenceville, GA, USA) media with 10% fetal bovine serum (FBS), containing penicillin/streptomycin, at 37 °C and in the presence of humidity and 5% CO₂.