ARPE-19 cells were obtained from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. ARPE-19 cells were grown in DMEM/F12 medium supplemented with 10% fetal bovine serum. For FK866 (Sigma-Aldrich; St Louis, MO, USA) treatment, ARPE-19 cells were serum starved overnight and treated with different doses (0.01–10 μmol/L) of FK866 as described previously.[15 (link)] Cells were collected at different time intervals (24, 48, and 72 h) after FK866 treatment and total RNA was extracted for the evaluation of hub gene expression.
Fk866
Manufactured by Merck Group
Sourced in United States, Germany
FK866 is a laboratory reagent used for research purposes. It functions as an inhibitor of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), which is involved in the biosynthesis of nicotinamide adenine dinucleotide (NAD+).
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Cellevent caspase 3 7 green detection reagent, by Thermo Fisher Scientific (3 mentions)
Olaparib, by Selleck Chemicals (2 mentions)
Ex 527, by Merck Group (2 mentions)
Fk866, by Selleck Chemicals (2 mentions)
Stf118804, by Bio-Techne (2 mentions)
Nicotinamide, by Merck Group (2 mentions)
Nad nadh quantitation kit, by Merck Group (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Opti mem medium, by Thermo Fisher Scientific (2 mentions)
U0126, by Merck Group (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Gmx1778, by Merck Group (2 mentions)
Puromycin, by Thermo Fisher Scientific (2 mentions)
Nicotinamide riboside, by ChromaDex (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Fk866, by Cayman Chemical (2 mentions)
Etoposide, by Merck Group (2 mentions)
62 protocols using fk866
1
ARPE-19 Cells: FK866 Dose and Time Response
2
Modeling Alzheimer's Disease in Mice
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In total, 18 male APP/PS1 transgenic mice (age: 24 weeks; weight: 26±3 g) and 6 male C57BL/6 mice (age: 24 weeks; weight: 27±2 g) were used in the present study. The APP/PS1 mouse strain is a double-transgenic hemizygote that expresses a chimeric mouse/human amyloid precursor protein and mutant human presenilin-1. These transgenic mice were used as they develop behavioral and pathology features of AD similar to patients and have been used in previous AD studies worldwide (17 (link)–19 (link)). Separately, C57BL/6 mice were used as age-matched controls. The APP/PS1 transgenic mice were purchased from the Model Animal Research Center of Nanjing University, while C57BL/6 mice were obtained from the Experimental Animal Center of Shanghai Academy. The NAD+ group were intraperitoneally injected with NAD+ (30 mg/kg, Sigma-Aldrich; Merck KGaA) at 20 weeks of age and once every other day for 4 weeks. The FK866 (NAMPT inhibitor) group were intraperitoneally injected with FK866 (1 mg/kg, Sigma-Aldrich; Merck KGaA) at 20 weeks of age and once every other day for 4 weeks. The mice were housed in a controlled specific-pathogen-free environment (22±3°C; 60% relative humidity; 12-h light/dark cycle) with ad libitum access to water and food.
3
Optimizing FK866 for Senescence Modulation
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To determine the optimal concentration of FK866 without inducing robust cellular toxicity, 3,000 cells were seeded in 96-well plates and incubated in a humidified incubator at 37 °C for 24 h. Thereafter, the cells were treated with different concentrations of FK866 (0–100 nM; Sigma-Aldrich) for 72 h before the addition of CCK8 solution (10 μL/well). After incubation at 37 °C for 1 h, the absorbance at 450 nm was measured with a microplate reader. Subsequently, senescent LP MSCs were cultured in the presence or absence of different exogenous NAD intermediates, including 100 μM nicotinamide (NAM), 100 μM nicotinamide mononucleotide (NMN), 100 μM NAD, and 5 μM of Sirt1 activator resveratrol (RSV) for 48 h. For FK866-induced cellular senescence, young EP cells were pre-treated with complete medium containing 10 nM FK866 or Vehicle (DMSO) for 24 h, and then the cells following FK866 treatment were respectively exposed to 100 μM NAM, 100 μM NMN, 100 μM NAD, and 5 μM RSV for 48 h. Afterward, cells were collected for SA-β-gal assay, NAD+ and NAD+/NADH measurement, Sirt1 activity assay, and protein expression studies.
4
Femur Fracture Model in Mice
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8-Week-old mice were anesthetized by isoflurane inhalation anesthesia. The right femur was then shaved and scrubbed with betadine. All instruments and pin implants were sterilized before use. A 1 cm surgical incision was made over the anterolateral distal femur to expose the mid-point femur. A mid-shaft transverse fracture was made using a sharp scalpel. A 24-gauge stainless-steel pin was passed into the intramedullary canal to stabilize the fracture with the keen flexed. Radiographs were taken immediately after surgery and before sacrifice to confirm pin placement and fracture pattern. Animals were given buprenorphine (0.05 mg/kg) to alleviate any surgical pain. Sutures were checked daily for 3 days and removed on day 7.
Mice were randomly allocated into either vehicle (DMSO) or FK866 group and received intraperitoneal (IP) injections of FK866 (10 mg/kg, Sigma-Aldrich, USA) or vehicle in pyrogen-free saline every 12 h from the first postoperative day. On day 21, mice were euthanized and prepared for analysis.
Mice were randomly allocated into either vehicle (DMSO) or FK866 group and received intraperitoneal (IP) injections of FK866 (10 mg/kg, Sigma-Aldrich, USA) or vehicle in pyrogen-free saline every 12 h from the first postoperative day. On day 21, mice were euthanized and prepared for analysis.
5
Chemosensitivity Screening of FK866
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Cells were screened for their chemosensitivity to FK866 (Sigma-Aldrich, France). They were grown in SFDM without nicotinamide for 72 h. Five thousand cells per well were plated in 96-well plates in SFDM medium without nicotinamide. Twenty-four hours later the media was supplemented with increasing concentrations of FK866 and incubated for an additional 72 h period. Each experiment was done in triplicate and repeated at least two times. These cells were treated for 72 h with increasing concentrations of FK866 ranging from 0 to 1000 nM (in a progression by a factor4). FK866 was next tested at a given concentration in association with increasing concentrations of gemcitabine, oxaliplatin or 5FU (0 to 1000 μM).
6
Metabolic Profiling of Cancer Cells
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Carboplatin was provided by the university hospital Leuven (UZL), who purchased it from Hospira. NAC, αKB, FK866 (Daporinad), L-13C5-glutamine and L-13C3-serine were obtained from Sigma-Aldrich, whereas D-glucose 13C6 was purchased from Cambridge Isotope Laboratories. Olaparib (AZD2281) was purchased from Selleckchem. All ingredients to make fresh RPMI were purchased from Sigma-Aldrich. Hygromycin B was purchased from Invitrogen, G418 sulfate (Geneticin) and puromycin were purchased from Gibco.
7
GSPE Proanthocyanidin Purity Protocol
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The GSPE with over 98% proanthocyanindin purity was purchased from Rongsheng Biotechnology Co., Ltd. (Cat No.: 84929–27-1; Xi’an, China) and dissolved in drinking pure water for in-vivo study and in DMSO (Cat No.: C6164, Signa, USA) for in-vitro treatment. NAMPT inhibitor, Fk866 (Cat No.: F8557), and SIRT1 inhibitor, EX-527 (Cat No.: E7034), were purchased from Sigma (Sigma-Aldrich, USA). The NAD+ precursor, NMN with a purity of over 99.5% was purchased from Hygieia Biotechnology Co., Ltd (Cat No.: XJY01210128, Shenzhen, China).
8
Oxidative stress induction protocol
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Two days before stimulation, cells were seeded in 6-, 24-, or 96-well culture plates (2 × 105, 5 × 104 or 1 × 104 cells/well, respectively). 1.5 h prior to stimulation, the culture medium was replaced with 2 mL, 500 μL or 100 μL of fresh medium, respectively. DPQ (Cayman, Cat#14450, 50 μM), FK866 (Sigma, Cat#F8557, 10 nM), NR (Carbosynth, Cat#NN15702, 1 mM) or vehicle control (DMSO ( ≥ 99.5% purity: Sigma, Cat#D5879) or H2O) was administered at the same time. DPQ and FK866 were diluted with DMSO to 50 mM and 10 μM, respectively. NR was diluted with H2O to 1 M. H2O2 (FUJIFILM Wako, Cat# 081-04215) was diluted to 100× the final concentration with H2O, and the diluted H2O2 was added to the culture medium at 20, 5 or 1 μL/well for 6-, 24-, or 96-well culture plates, respectively.
9
Modulating PPM1D in Astrocytes
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PPM1Dtrnc. astrocytes were treated with 50 μg/mL cycloheximide or 10 μM MG132 (both Sigma) for the indicated amount of time. Cells were then washed, pelleted, and lyzed for subsequent immunoblotting approaches, as described above. Quantification of immunoblot intensity was calculated using ImageJ software, and consisted of multiple (n = 3) blots. Irradiation of cells was performed using an X-RAD KV irradiator (Precision X-ray), and treatment consisted of an unfractionated, 10 Gy dose. PPM1D inhibitor treatment with GSK2830371 (Selleckchem), consisted of 50 nM treatment, 24 h prior to IR. FK866 (Selleckchem), GPP78 (Tocris Bioscience), STF118804 (Tocris Bioscience), STF31 (Tocris Bioscience), 5-azacytidine (Selleckchem), and DCT (Selleckchem) were dissolved in DMSO and used for treatment as indicated. nicotinamide riboside (ChromaDex Inc.) and nicotinamide (Sigma) were dissolved in water while nicotinic acid (Sigma) was dissolved in PBS, prior to treatment alone or in combination with FK866, as indicated.
10
Cell Line Maintenance and Antibody Use
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HEK293 cells, SKOV3 cells, OVCAR-3, and ES-2 cells were all maintained in regular DMEM supplemented with 10% (v/v) FBS, penicillin-streptomycin. The antibodies applied in this study include anti-HA antibody (Beyotime, AF0039), anti-Flag antibody (Sigma F1804), anti-CtBP1 antibody (BD biosciences 612042), anti-CtBP2 antibody (BD biosciences, 612044), anti-γH2AX (Sigma-Aldrich, SAB5600038) and normal mouse IgG (Santa Cruz, sc2025). The chemicals include MTOB (4-methylthio-2-oxobutanoate, Sigma-Aldrich, k6000), 2-DG (2-Deoxy-D-glucose, Sigma-Aldrich, D8375), 3-BP (3-bromopyruvate, Sigma-Aldrich, 16490), FK866 (Sigma-Aldrich, F8557), cisplatin (Sigma-Aldrich, PHR1624), metformin (Sigma-Aldrich, PHR1084), penicillin-streptomycin (ThermoFisher), Puromycin (ThermoFisher). Luciferin (Yeasen), Doxycycline (Beyotime).
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