A split luciferase assay was conducted in accordance with the protocol provided by Promega Corporation (WI, USA). HEK293T cells were transfected with V5_LAR_FRBLgBiT/Myc_LAR_FKBPSmBiT-expressing plasmids or control V5_LAR_LgBiT/Myc_LAR_SmBiT-expressing plasmids so that the total DNA amount was 100 ng/well. After transfection, the cells were cultured at 37°C under 5% CO2 for 20∼24 h. Then, before application of the substrates, the culture media were exchanged with Opti-MEM® I (1X) (Gibco® by Life TechnologiesTM, Thermo Fisher Scientific) and incubated at ambient temperature for 10 min. The transfected wells were recorded for at least 5 min before substrate addition to assess the basal status. Luciferase substrate (Promega Corporation) was applied for 5 min before the generated luminescence was recorded for 40 counts. Then, vehicle or rapamycin (20 nM) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) was applied, and the subsequent changes in luminescence were recorded for 61 counts. The interval between counts was 1 min. The whole process was performed at ambient temperature. Luminescence was detected using a Centro XS3 LB960 High Sensitivity Microplate Luminometer (Berthold Technologies, Bad Wildbad, Germany).
Rapamycin
Manufactured by Fujifilm
Sourced in Japan, United States
Rapamycin is a laboratory product manufactured by Fujifilm. It is a chemical compound with a well-defined molecular structure and function. Rapamycin's core function is to act as an immunosuppressant and anti-cancer agent in various research and scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Ly294002, by Fujifilm (2 mentions)
Luciferase substrate, by Promega (1 mentions)
Opti mem 1 1x, by Thermo Fisher Scientific (1 mentions)
Centro xs3 lb960 high sensitivity microplate luminometer, by Berthold Technologies (1 mentions)
Ku 0063794, by Fujifilm (1 mentions)
Benchmark microplate reader, by Bio-Rad (1 mentions)
Roscovitine, by Merck Group (1 mentions)
Bx795, by Enzo Life Sciences (1 mentions)
Pp242, by Cayman Chemical (1 mentions)
Wortmannin, by LC Laboratories (1 mentions)
Prism 7j, by GraphPad (1 mentions)
Wst 8 kit, by Nacalai Tesque (1 mentions)
Lipopolysaccharides (lps), by Fujifilm (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Anti tgf β antibody, by R&D Systems (1 mentions)
Normal mouse igg, by Fujifilm (1 mentions)
Celltiter 96 non radioactive cell proliferation assay, by Promega (1 mentions)
19 protocols using rapamycin
1
Luminescence-based Dimerization Assay
2
Evaluating Anti-CD146 Antiserum and mTOR Inhibitors on Cell Viability
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Cells were plated in normal growth medium in 96-well cell culture plate (2000 cells per well). After 6 h, the medium was exchanged. To determine the effect of antiserum, inactivated antiserum against CD146 or normal rabbit serum (Life Technologies) was added to culture medium for adjusting to a final concentration of 5% or 10% (v/v). To determine the effect of mTOR inhibitors, various concentrations of rapamycin (Wako) and Ku-0063794 (Wako) were added to culture medium. Cell viability was measured every 24 h for 4 days by WST-8 assay using the Cell Counting Kit-8 (Dojin, Kumamoto, Japan) on a Benchmark Microplate Reader (Bio-Rad, Hercules, CA, USA).
3
Inhibition of Cellular Kinases in Starfish Oocytes
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Wortmannin (LC Laboratories), pp242 (Cayman Chemical), BX795 (Enzo Life Sciences), and roscovitine (Merck) were dissolved in DMSO at 20 mM as a stock solution, and used at final concentrations of 40, 2, 6, and 30 µM, respectively. Rapamycin (FUJIFILM Wako Pure Chemical) was dissolved in DMSO at 20 mM as a stock solution. It was used at a final concentration of 20 µM, a concentration that was previously shown to be sufficient for TORC1 inhibition in starfish oocytes (Hiraoka et al., 2011 (link)).
4
Evaluating JPH203 and Rapamycin Effects on Cell Proliferation
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Inhibitory effects of JPH203 (MedKoo, Morrisville, NC) on the proliferation activities of Caki-1, ACTN and HEK293 cells were evaluated using a WST-8 kit (Nacalai Tesque, Kyoto, Japan) according to the manufacturer’s protocol. The experiment was performed as previously reported35 (link). IC50, which is the concentration at which the cell viability is reduced by half, was calculated using GraphPad Prism 7J (GraphPad Software, La Jolla, CA). Similarly, inhibitory effects of JPH203 and rapamycin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) on the proliferation activities of Caki-1 and ACTN cells were also evaluated using a WST-8 kit. The cells were seeded onto 96-well plates (0.5 × 104 cells/well) and cultured with JPH203 (10 or 30 µM), rapamycin (10 or 30 nM), or DMSO (0.5%, a vehicle control), for three days, after which a viability assay was performed.
6
Cardiomyoblast Cell Culture and Treatment
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Rat cardiomyoblast cells (H9c2) were purchased from the European Collection of Authenticated Cell Cultures (ECACC) and maintained in Dulbecco's modified Eagle's medium (DMEM; Wako, Tokyo, Japan) supplemented with 10% fetal bovine serum and 100 U·mL−1 penicillin/streptomycin (both from Sigma‐Aldrich, St. Louis, MO, USA) at 37 °C and 5% CO2. For all experiments, cells were serum‐starved overnight. LY294002 (Cell Signaling Technology, Danvers, MA, USA) and rapamycin (Wako) were added 1 h before the treatment of HDL.
7
Progranulin regulation of cell proliferation
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The progranulin siRNA or control siRNA-transfected cells were seeded in 96 well plates at a density of 3.5 × 10ˆ5 cells/mL. Proliferation was determined using the CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega, WI, USA). Anti-human progranulin mouse monoclonal antibody 6B3 was kindly provided from Dr. Ginette Serrero (A&G Pharmaceutical, MD, USA). Normal mouse IgG (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was used as control. The cells were seeded in 96 well plates at the density of 5.0 × 10ˆ4 cells/mL with anti-progranulin antibody or Normal mouse IgG (200 μg/mL), and proliferation was determined based on the above-mentioned description. To regulate the signaling pathway, the cells were cultured with the combination of rapamycin (20 nM) (FUJIFILM Wako Pure Chemical, Osaka, Japan), chloroquine (20 nM), Z-VAD-FMK (50 μM) (MBL, Aichi, Japan), LY2109761 (250 μM) (ChemScene, NJ, USA), or anti-TGF-β antibody (2 μg/mL) (R&D Systems, MN, USA).
8
Effect of mTOR Inhibitors on TGF-β2-Induced TM Cells
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The hTM cells from three donor eyes (46 years old, 52 years old, and 55 years old, without glaucoma) were used in this study, and hTM cells were cultured in growth medium containing 10% fetal bovine serum (FBS) and Antibiotic Antimycotic Solution (100 ×) (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in 5% CO2. Cells were seeded at 5000 cells/cm2 and grown to confluence. Cells from passages 3–6 were used in the experiments. The cells were treated with TGF-β2 for 24 h with or without pretreatment for 30 min with rapamycin (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) or Torin 1 (Selleck Chemicals, Houston, TX, USA) as an mTOR inhibitor. This study is not deemed a Human Subject Research because cells were acquired post-mortem from de-identified donor tissues and is thus considered exempt by University of Tokyo’s Institutional Review Board (IRB). Nevertheless, all experiments were done in accordance with the tenets of the Declaration of Helsinki. And this study does not include in vivo data, but all the in vitro studies were performed in accordance with ARRIVE guideline.
9
Regulation of Cell Cycle Progression
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The sources for the following chemicals, reagents and antibodies were: Rapamycin (WAKO Chemical, Richmond, VA, USA); Torin 2 and Ku0063794 (TOCRIS, Bristol, UK); IGF-1 (BioVision, Milpitas, CA, USA); DHT, LY294002, SP600125, SB202190, PD153035, and PD98059 (Sigma Aldrich, St. Louis, MO, USA); characterized fetal bovine serum (FBS) (ATCC, Manassas, VA, USA); anti-HMMR (Bethyl, Montgomery, TX, USA); anti-p-Rb(S807/811), anti-CDK2, anti-E-cadherin (24E10), anti-vimentin (D21H3), anti-p-Erk1/2 (T202/Y204), and anti-Erk (Cell signaling, Danvers, MA, USA); anti-cyclin D2 (Santa Cruz, Dallas, TX, USA); anti-GAPDH (Bioworld Technology, St. Louis Park, MN, USA); anti-β-actin, anti-FlagM2, and dextran-charcoal stripped fetal bovine serum (DC-FBS) (Sigma Aldrich); characterized fetal bovine serum (FBS) (HyClone, Logan, UT, USA).
10
Autophagy and Apoptosis Induction Assays
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BTZ and RCS were purchased from Selleck Chemicals, and were dissolved in dimethyl sulfoxide (DMSO; Nacalai Tesque, Inc.) to generate stock solutions (BTZ, 1 mM; RCS, 10 mM). Z-VAD-fmk, a pan-caspase inhibitor, was purchased from Peptide Institute, Inc. 3-Methyladenine (3-MA) was obtained from Tokyo Chemical Industry Co., Ltd. Necrostatin-1, a specific inhibitor of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), was purchased from Enzo Life Sciences, Inc. Cycloheximide (CHX) was purchased from Calbiochem. N-acetyl-L-cysteine (NAC). Bafilomycin A1, rapamycin, puromycin dihydrochloride, recombinant human TNF-α and staurosporine were purchased from FUJIFILM Wako Pure Chemical. Thapsigargin (TPG) was purchased from Nacalai Tesque, Inc.
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