Rad001

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).

QGP-1, a human pNET cell line, was purchased from Japanese Collection of Research Bioresources (JCRB, Tokyo, Japan). NIT-1, a mouse pNET cell line, was purchased from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). The passage number of the two cell lines used in this study was less than 15. The QGP-1 cell line that we used was sent to the Center for Genomic Medicine of National Cheng Kung University for genotyping on Jun 2016, and the result showed the same STR DNA profile as those in the JCRB database. QGP-1 and NIT-1 cells were cultured in RPMI-1640 medium (Hyclone, South Logan, Utah, USA) and F12 Kaighn’s medium (Gibco, Grand Island, NY, USA), respectively, containing 10% fetal calf serum and antibiotics. The shRNAs targeting PCK2 and Pck2 were obtained from the National RNAi Core Facility of Academic Sinica (Taipei, Taiwan). Rapamycin and RAD001 were purchased from LC laboratories (Woburn, MA, USA) and Selleckchem (Munich, Germany), respectively. 2-Deoxy-D-glucose (2-DG) was purchased from Seahorse Bioscience (Billerica, MA, US). The primary antibody against PEPCK-M was purchased from GeneTex (GTX114919, USA). Antibodies against PEPCK-C (sc-74825) and GAPDH were purchased from Santa Cruz (Texas, US). Antibody against β-actin was purchased from Millipore (MA, US).

Human RCC cell lines, characterized as an adenocarcinoma (ACHN) and clear cell carcinomas (Caki-1 and 769-P), were originally purchased from ATCC [25 –27 (link)]. All cells were cultured in RPMI with 10% FBS, 1% glutamine, and 1% Pen/Strep. For in vitro experiments, cells were seeded on the appropriated plates overnight and treated with HCQ (75 or 100 μM, Acro Chemicals), RAD001 (10 μM, LC Laboratories), bafilomycin A1 (50 nM, Sigma), or spautin-1 (10 μM, Sigma) for 48 hours. Bortezomib (Velcade, 1 μ g/ml) was obtained from the Fox Chase Cancer Center pharmacy and treated cells for 16 hours.

The reagents used and their preparation were as follows: RAD001 (LC laboratories, USA) was dissolved in DMSO, then diluted with PBS to a stock solution of 10 mM and stored at −20 °C. Torin1 (Tocris, Bristol, UK) was dissolved in DMSO to yield a stock solution of 1 mM. Chloroquine (Sigma-Aldrich Ltd., Rehovot, Israel) was diluted in PBS to a concentration of 10 mM. The controls were treated with the same medium as that diluting the drugs.