HepG2 cells (5 × 103 cells/96-well plate) were incubated for 24 hours, and starvation was performed for 4 hours. Uridine, N-acetylmannosamine, cinobufagin, and cinnamoylglycine were purchased from Sigma (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). HepG2 cells were treated with different concentrations of Uridine, N-acetylmannosamine, cinobufagin, and cinnamoylglycine for 24 hours, and then incubated with 3-(4, 5-dimethyl-2-thiazolyl)−2, 5-diphenyltetrazolium bromide (MTT, Sigma Aldrich) for 4 hours at 37°C and 5% Co2 incubator. The formazan crystals were dissolved in DMSO and their absorbance was measured at 570 nm using a microplate reader (Molecular Devices, Silicon Valley, CA, USA).
Cinobufagin
Manufactured by Merck Group
Sourced in United States
Cinobufagin is a laboratory reagent produced by Merck Group. It is a bioactive compound derived from the skin and parotid glands of certain toad species. Cinobufagin is used in various research applications, including the study of cell signaling pathways and the development of potential therapeutic agents.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Cinobufagin, by Yuanye Bio-Technology (2 mentions)
Beas 2b bronchial epithelial cell lines, by American Type Culture Collection (2 mentions)
Bufalin, by Merck Group (2 mentions)
Rpmi 1640, by Keygen Biotech (1 mentions)
Formic acid, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Keygen Biotech (1 mentions)
Cinnamoylglycine, by Merck Group (1 mentions)
Uridine, by Merck Group (1 mentions)
N acetylmannosamine, by Merck Group (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Microplate reader, by Molecular Devices (1 mentions)
Methyl alcohol, by Thermo Fisher Scientific (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
High glucose dulbecco s modified eagle s medium dmem, by Cytiva (1 mentions)
Pentobarbital sodium, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp labeled secondary antibody, by Cell Signaling Technology (1 mentions)
7 protocols using cinobufagin
1
Evaluating Cytotoxicity of Bioactive Compounds
2
Synthesis and Characterization of BF211 Nanoparticles
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BF211 was synthesized in our laboratory as previously described.4 (link) Distearoyl phosphoethanolamine-PEG2000 (2000PEG-DSPE), cholesterol (CH), hydrogenated soybean phospholipids (HSPC) were obtained from Lipoid GmbH (Ludwigshafen, Germany). Ammonium sulfate was purchased from Nanjing Chemical Reagent Company (Nanjing, China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), 4% paraformaldehyde (PFA), 4´,6-diamidino-2-phenylindole (DAPI), DiD, DiR, DiI, PKH-26, trypsin-EDTA solution, DMEM and RPMI 1640 were supplied by KeyGEN BioTECH Co. Ltd (Nanjing, China). Creatine Kinase (CK) kit and lactate dehydrogenase (LDH) kit, Doxorubicin (Dox) were purchased from Nanjing JinYibai Biological Technology Co. Ltd. (Nanjing, China). Cinobufagin was supplied by Sigma Corporation (St. Louis, MO, USA). Poloxamer 188 (P188) (PEO/PPO/PEO ratio is 80/27/80, MW 8600) and Tween 80 for injection were purchased from Feiyu BioTECH Co. Ltd (Nanjing, China). HPLC-grade methanol (MeOH), acetonitrile (ACN) and formic acid were obtained from Merck (Darmstadt, Germany). Deionized (DI) water produced by a Milli-Q® Plus System (Billerica, MA, USA) with a resistivity of 18.2 mΩ·cm-1 was used in all the experiments. All other chemicals and reagents were obtained from Sigma Aldrich (Shanghai, China), and used without further purification.
3
Cinobufagin Cytotoxicity Evaluation
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Cinobufagin was purchased from Yuanye Biotechnology (Shanghai, China). Stock solution of Cinobufagin was prepared in 100% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) and then diluted in complete cell culture medium to make working solutions. The same solutions without Cinobufagin were used as vehicle controls.
Human SW480, SW1116 colorectal adenocarcinoma and BEAS-2B bronchial epithelial cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and the human L-O2 liver and NCM460 colon epithelial cell lines were purchased from KenGen (Nanjing, China). All other cell lines were bought from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, ThermoFisher Scientific, Shanghai, China) containing 10% fetal bovine serum (FBS) (Gibco, ThermoFisher Scientific, Shanghai, China). The cells were maintained at 37 °C in a humidified incubator with 5% CO2, following instructions from the providers. Cell authenticity was confirmed by short tandem repeats (STR) profiling.
Human SW480, SW1116 colorectal adenocarcinoma and BEAS-2B bronchial epithelial cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and the human L-O2 liver and NCM460 colon epithelial cell lines were purchased from KenGen (Nanjing, China). All other cell lines were bought from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, ThermoFisher Scientific, Shanghai, China) containing 10% fetal bovine serum (FBS) (Gibco, ThermoFisher Scientific, Shanghai, China). The cells were maintained at 37 °C in a humidified incubator with 5% CO2, following instructions from the providers. Cell authenticity was confirmed by short tandem repeats (STR) profiling.
4
Cytotoxicity Evaluation of Bufalin and Cinobufagin
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Bufalin, Cinobufagin, bovine serum albumin (BSA), 3-(4, 5-dimethyl-2-thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT), glutaraldehyde and dimethyl sul-foxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO, USA). Acetonitrile and methyl alcohol were purchased from Fisher Scientific (Pittsburgh, PA, USA). High-glucose Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine serum (FBS), Pancreatic enzyme and Ethylene Diamine Tetraacetic Acid (EDTA) were purchased from Hyclone Laboratories (Logan, UT, USA). Adriamycin was purchased from Wanle Pharmacia (Shenzhen, China). Pentobarbital sodium was obtained from Merck Corp (Darmstadt, Germany). All solvents were of high-performance liquid chromatogra-phy grade. All reagents were of analytical grade.
5
Cinobufagin and Cisplatin Combination Therapy
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Cinobufagin and CDDP were purchased from from Sigma (St. Louis, MO, USA). All media were obtained from GE Healthcare (Hyclone, UT, USA). Fetal bovine serum was from Gibco (Australia). Cell Counting Kit-8 (CCK-8) was obtained from Dojindo (Kumamoto, Japan). Primary antibody for Notch-1 intracellular domain (NICD1), Hes1, VEGF, Bcl-2 and Bax were from Abcam (Cambridge, UK). RIPA Lysis Buffer, primary antibodies against, Cleaved Caspase-9, Cleaved Caspase-3, MMP-2, MMP-9 and horseradish peroxidase (HRP)-labeled secondary antibody were from Cell Signaling Technology (Beverly, MA, USA). Amersham ECL Western blotting detection reagents and analysis system was purchased from GE Healthcare (Hyclone, UT, USA) and all other chemicals were from Sigma (St. Louis, MO, USA), unless otherwise indicated.
6
High-Throughput Screening of Small Molecules
7
Cinobufagin Cytotoxicity Screening on Cell Lines
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Cinobufagin was purchased from Yuanye Biotechnology (Shanghai, China). Stock solution of Cinobufagin was prepared in 100% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) and then diluted in complete cell culture medium to make working solutions. The same solutions without Cinobufagin were used as vehicle controls.
Human SW480, SW1116 colorectal adenocarcinoma and BEAS-2B bronchial epithelial cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and the human L-O2 liver and NCM460 colon epithelial cell lines were purchased from KenGen (Nanjing, China). All other cell lines were bought from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Dulbecco's Modi ed Eagle's Medium (DMEM, Gibco, Life Technologies Corporation) containing 10 % fetal bovine serum (FBS, Kangyuan, China). The cells were maintained at 37°C in a humidi ed incubator with 5% CO2, following instructions from the providers. Cell authenticity was con rmed by short tandem repeats (STR) pro ling.
Human SW480, SW1116 colorectal adenocarcinoma and BEAS-2B bronchial epithelial cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and the human L-O2 liver and NCM460 colon epithelial cell lines were purchased from KenGen (Nanjing, China). All other cell lines were bought from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in Dulbecco's Modi ed Eagle's Medium (DMEM, Gibco, Life Technologies Corporation) containing 10 % fetal bovine serum (FBS, Kangyuan, China). The cells were maintained at 37°C in a humidi ed incubator with 5% CO2, following instructions from the providers. Cell authenticity was con rmed by short tandem repeats (STR) pro ling.
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