Fk866

The inhibitor FK866 (cat # S2799) and NMN (cat # S5259) were obtained from Selleck Chemicals. LoVo and RKO cells were seeded into 96-well culture plates at 1000 cells/well and were allowed to attach for 24 h in an incubator before treatment with FK866 or NMN.

MEFs (WT and p32KO) were obtained in our laboratory (Yagi et al, 2012). All cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM; 1,000 mg/l glucose; Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% FBS at 37°C in a humidified atmosphere with 5% CO₂. MEFs were incubated with the Nampt inhibitor, FK866 (10 nM; Selleck Chemicals Llc, Houston, TX, USA), for 24–48 h in 5% CO₂ at 37°C, followed by measurement of total cellular NAD⁺ and NADH content using the NAD/NADH‐Glo™ Assay (Promega). YM201636 (13576) was purchased from Cayman Chemical. These compounds were solubilized in DMSO (Sigma–Aldrich, D8418), which was used as vehicle control.

All experiments were performed in accordance with the rules of the in vivo ethical committee of University of Montreal (CDEA #17-103) and CRCHUM (C18046GFs). For this study, 6–7-week old female nude mice (Hsd:Athymic Nude- Foxn1nu, Envigo) were implanted subcutaneously with 750,000 KP4 or 1,000,000 PANC-1 cells in 100 μL of 20% Matrigel (Corning) in saline. Tumor volume was determined by using a caliper, following the formula 4/3*π*(L*W*T), where L represents the length of the tumor, W the width, and T the thickness (all measured in millimeters). For in vivo experiments, metformin was purchased from Sigma-Aldrich (PHR1084) and FK866 from Selleckchem (S2799). The compounds were dissolved in the vehicle: 45% Propyleneglycol (Sigma-Aldrich) + 5% Tween 80 (Sigma-Aldrich) + ddH2O. Mice were injected intraperitoneally (with 100 μL) 5 days per week, starting on day 11 post-engraftment, with either 75 mg/kg/d metformin, 20 mg/kg/d FK866, metformin and FK866 combined (Met+FK; 75 mg/kg/d and 20 mg/kg/d, respectively), or vehicle for 4 weeks.

The anaplastic meningioma cell line (CRL-3370) was obtained from the American Type Culture Collection. The cell culture medium consisted of DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, USA). The cell line was maintained at 37 °C in a humidified incubator with 5% CO₂.
Cell viability was determined using Cell Counting Kit-8 (CCK-8) (Biosharp, China, BS350B). Briefly, 5mg FK866 (Selleck, USA, S2799) was dissolved in 1.2771 ml dimethyl sulfoxide (DMSO) (Sigma, USA, D2650), then 10 mM FK866 was obtained. Then 10 mM FK866 was diluted with DMSO to obtain 10 μM FK866. After incubation with DMSO or different concentration of FK866 (25μM, 12.5μM, 2.5μM, 0.01μM, 0.005μM) for 24h, 48h or 72 h, 10 µl of CCK-8 reagent was added to each well, and the plates were further incubated in an incubator for 2h. Subsequently, the absorbance was measured at 450 nm by a microplate reader (Biotex, Synergy H1, USA). The cell viability (%) was calculated as follows: (average OD of treated groups at 24, 48 or 72 h/average OD of untreated groups at the same time point) x 100%.

SH-SY5Y and U937 cell lines were obtained from the Beijing Stem Cell Bank, Chinese Academy of Sciences (Beijing, China). All other cell lines were purchased from the American Type Culture Collection (Manassas, VA, United States). MDA-MB-231, BT549, MDA-MB-468 and MCF7 cells were cultured in DMEM (HyClone; GE Healthcare, Chicago, IL, United States) containing 10% fetal bovine serum (FBS; PAN-Seratech GmbH, Aidenbach, Germany). HCC1937 and U937 cells were cultured in RPMI medium (HyClone; GE Healthcare) containing 10% FBS. MCF10A cells were cultured using the MEGM Bullet kit (Lonza Group, Basel, Switzerland) and 10 ng/ml cholera toxin. SH-SY5Y cells were cultured in DMEM/F12 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, United States) containing 10% FBS. All cell lines were propagated at 37˚C with 95% humidity in a 5% CO₂ incubator.
Dezocine was obtained from Yangzi River Pharmaceuticals Group (Taizhou, Jiangsu, China). FK866 and NMN were purchased from Selleck Chemicals LLC, Houston, TX, United States). Morphine was obtained from Northeast Pharmaceutical Group Shenyang No.1 Pharmaceutical Co. Ltd (Shenyang, Liaoning, China). Morphine, dezocine and NMN were dissolved in ddH₂O. FK866 was dissolved in ethanol. All compounds were diluted in appropriate media for cell culture studies.