CAP, doxycycline, and actinonin were obtained from Wako Pure Chemicals, Osaka, Japan. Antibodies against ATF4, p-eIF2α Ser51 (3795S), and eIF2α (9722S) were purchased from Cell Signaling Technology (Danvers, MA, U.S.A.). Anti-COXI (Ab14705) was from Abcam, Cambridge, U.K. COXIII and SDHA antibodies were from Thermo Fisher Scientific, Waltham, MA, U.S.A. The secondary antibodies, anti-mouse horseradish peroxidase (HRP)-conjugated IgG (#7076) and anti-rabbit HRP-conjugated IgG (#7074), were from Cell Signaling Technology.
Doxycycline
Manufactured by Fujifilm
Sourced in Japan
Doxycycline is a broad-spectrum antibiotic medication used in a variety of laboratory applications. It is a tetracycline derivative that inhibits bacterial protein synthesis. Doxycycline can be used as a selective agent in cell culture to maintain transgene expression or induce gene expression in inducible systems.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Eif2α 9722, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
Sodium pyruvate, by Nacalai Tesque (1 mentions)
Non essential amino acids (neaa), by Nacalai Tesque (1 mentions)
Heat inactivated fetal bovine serum, by Biowest (1 mentions)
Doxycycline, by Nacalai Tesque (1 mentions)
Cellcarrier 96, by PerkinElmer (1 mentions)
Cellcarrier 384 ultra microplate, by PerkinElmer (1 mentions)
Rock inhibitor, by Fujifilm (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
Imatrix 511, by Nippi (1 mentions)
Stemfit medium, by Ajinomoto (1 mentions)
Doxycycline, by Takara Bio (1 mentions)
Hygromycin b, by Fujifilm (1 mentions)
Ptet off advanced vector, by Takara Bio (1 mentions)
Non essential amino acids (neaa), by Fujifilm (1 mentions)
Pd 0325901, by Adooq (1 mentions)
Chir 99021, by Focus Biomolecules (1 mentions)
9 protocols using doxycycline
1
Mitochondrial Stress Response Regulation
2
Doxycycline-Induced RCAS1 Overexpression Model
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To investigate the biological functions of RCAS1, a doxycycline (Dox)-induced RCAS1 overexpression model was established in murine fibroblast L cells (L/ind RCAS1) as described in a previous report (27 (link)). The present study used the human uterine cervical adenocarcinoma cell line SiSo and L/ind RCAS1 cells. These cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Biowest), 100 nM non-essential amino acids (Nacalai Tesque, Inc.) and 1 mM sodium pyruvate (Nacalai Tesque, Inc.) at 37°C in a 5% CO2 atmosphere. To induce exogenous RCAS1 expression, doxycycline (Dox; FUJIFILM Wako Pure Chemical Corporation) was used. Dox was dissolved in dH2O at 1 mg/ml concentration and L/ind RCAS1 cells were treated with 0.5 µg/ml Dox for the indicated times.
3
Differentiation of Myocytes from iPS Cells
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Screening and mechanistic studies were performed on myocytes differentiated from the iPS cells generated as mentioned above. iPS cells were cultured and maintained as a feeder‐free culture on iMatrix‐511 (892012; Nippi, Tokyo, Japan) in StemFit medium (AK02N; Ajinomoto, Tokyo, Japan) as previously described 14 . Differentiation of MM iPS cells into myocytes was performed by the forced expression of MyoD1 under the control of doxycycline (D5897; Wako, Osaka, Japan) as previously described 12 , 15 . Briefly, on day 0, cells were plated on Matrigel‐coated CellCarrier‐96 or CellCarrier‐384 Ultra microplates (6057300; PerkinElmer, Waltham, MA) with Rock inhibitor (251‐00514; Wako); the next day, medium was switched to Primate ES Cell Medium. On day 2, doxycycline was added to the same medium, and the latter was switched to Minimum Essential Medium Eagle, Alpha Modification (α‐MEM) (21444‐05; Nakalai Tesque, Kyoto, Japan) containing 5% knockout serum replacement (10828028; Thermo Fisher Scientific, Waltham, MA) on day 3. The medium was changed every other day until day 8.
4
Inducible Gene Expression in LNCaP Cells
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LNCaP cells were transfected with pTet-Off Advanced vector (Clontech). A stable transformant was isolated by 800 μg/ml G418 (Wako), and tested for induction according to the manufacturer’s protocol. LNCaP tet-off cells were maintained in RPMI1640 medium with 10% FCS containing 400 μg/ml G418 and 1 μg/ml doxycycline (Clontech). LNCaP tet-off cells were transfected with pTRE2hyg-FLAG-AIbZIP, selected using 1 mg/ml hygromycin B (Wako), and maintained in RPMI 1640 medium with 10% FCS containing 400 μg/ml G418, 500 μg/ml hygromycin B and 1 μg/ml doxycycline.
5
Expansion of Murine Embryonic Stem Cells
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Every time a new batch of EBRTcPTbx6 cells was thawed, 1.5 μg/mL of puromycin was added for one week before propagation. EBRTcPTbx6 cells were maintained with Glasgow minimum essential medium (GMEM, Sigma–Aldrich), 10% FBS (MP Biomedicals), 0.1 mM NEAA (Wako), 1 mM sodium pyruvate (Sigma–Aldrich), 0.1 mM 2-mercaptoethanol (Sigma–Aldrich), 10 ng/mL doxycycline (Wako), 100 U/mL penicillin (Meiji Seika Pharma) and 100 μg/mL streptomycin (Meiji Seika Pharma), supplemented with 1000 U/mL mouse LIF (ORF genetics), 3 μM CHIR-99021 (FOCUS Biomolecules), and 0.4 μM PD-0325901 (Adooq Bio Science). 1 × 105 EBRTcPTbx6 cells were inoculated to a 60 mm cell culture dish (TrueLine, Nippon Genetics) that had been plated with MMC (Wako)-treated SNL 76/7 feeder cells (obtained from Riken Bioresource Center) on the previous day. The cells were passaged every other day, and subjected to isolation from feeder cells using differential adhesive properties to a gelatin-coated cell culture dish before performing differentiation assays as described below.
6
Feeder-free iPSC-derived Motor Neurons
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Motor neurons were generated from feeder-free iPSCs by transient expression of NIL factors using the tet-on inducible expression system. At the passage, dissociated iPSCs were seeded onto poly-L-ornithine (Merck) and Matrigel (Corning, Steuben County, NY, USA) coated culture plates and cultured in neuronal medium with the following composition: Neurobasal Plus medium (Thermo Fisher Scientific) containing 2% B27 Plus supplement (Thermo Fisher Scientific), 1% Culture One supplement (Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher Scientific), 200 μM L-ascorbic acid (Sigma), 200 μM dbcAMP (Nacalai), 20 ng/mL BDNF (Alomone labs), 20 ng/mL GDNF (Alomone labs), and 20 ng/mL NT-3 (Alomone labs). 20 μM Y27632, 1 μg/mL doxycycline (Wako), 1 μM retinoic acid (Merck), 1 μM purmorphamine (Merck) and 0.5 μg/mL iMatrix-511 silk were added only for 5 days. Half of the medium was changed every 3 or 4 days after day 5.
7
Curcumin Compound Preparation for Research
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Curcumin (Wako, Japan) stock solutions were prepared in sterile dimethyl sulfoxide (DMSO). The Curcumin analogue GO-Y030 was provided by Akita University, Japan. Its stock solution was prepared in sterile DMSO and stored at 4°C. Nile red (Wako, Japan) stock solution was prepared in ethanol and stored at 4°C. Doxycycline (Wako, Japan) was prepared in distilled water and stored at 4°C. Curcumin compounds and all dye solutions were kept in the dark to prevent light exposure.
8
Antibody Immunodetection Reagents
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The following antibodies were obtained commercially: anti-Rab11 rabbit polyclonal antibody (Invitrogen, Carlsbad, CA; #71-5300), which recognizes both Rab11A and Rab11B, anti-GM130 mouse monoclonal antibody (BD Biosciences, San Jose, CA; #610823), anti-EEA1 mouse monoclonal antibody (BD Biosciences; #610456), anti-LBPA mouse monoclonal antibody (Applied Biological Materials, Richmond, BC, Canada; #G043), anti-LAMP2 mouse monoclonal antibody (Thermo Fisher Scientific, Waltham, MA; #MA-28269), anti-TfR mouse monoclonal antibody (Invitrogen; #13-6800), anti-b-actin mouse monoclonal antibody (Applied Biological Materials; #G043), anti-ezrin mouse monoclonal antibody (Abcam, Cambridge, UK; #ab4069), horseradish peroxidase (HRP)-conjugated anti-GFP polyclonal antibody (MBL, Nagoya, Japan; #598-7), HRP-conjugated anti-mouse IgG goat polyclonal antibody (SouthernBiotech, Birmingham, AL; #1031-05), Alexa Fluor 555 + -conjugated anti-mouse IgG goat polyclonal antibody (Thermo Fisher Scientific; #A32727), and Alexa Fluor 555 + -conjugated anti-rabbit IgG goat polyclonal antibody (Thermo Fisher Scientific; #A32732). Other reagents used in this study were also obtained commercially: doxycycline (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and D/D solubilizer (Takara Bio, Shiga, Japan).
9
Pharmacological Compound Acquisition for Research
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Materials The following 47 compounds were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan for use in this study: acetylsalicylic acid, acyclovir, allopurinol, amitriptyline, amoxicillin, azathioprine, azithromycin, budesonide, caffeine, carbamazepine, chloramphenicol, chlorpheniramine maleate salt, chlorpromazine, diethylcarbamazine, digoxin, diphenhydramine hydrochloride, doxycycline, erythromycin, ethosuximide, fluconazole, folic acid, furosemide, haloperidol, hydrochlorothiazide, hydrocortisone, ibuprofen, isoniazid, loratadine, metformin, metoclopramide, metronidazole, nitrofurantoin, ondansetron, oseltamivir, paracetamol, phenytoin, potassium iodide, prednisolone, primaquine, propranolol, propylthiouracil, quinine hydrochloride, salbutamol, spironolactone, sulfamethoxazole, valproic acid and vancomycin.
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