Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone), oxacillin, and tetracycline were obtained from Sigma-Aldrich (Prague, CZ). DMSO (Penta, Prague, CZ), ethanol (Sigma-Aldrich, Prague, CZ), and deionized water were used as solvents for the preparation of stock solutions of antibiotics and Embelin (at 100 times higher concentration than the highest concentration tested). Methanol and trifluoroacetic acid (TFA), used as the mobile phase in HPLC assay, were purchased from Lachner (Neratovice, CZ) and Sigma-Aldrich (Prague, CZ), respectively.
Embelin
Manufactured by Merck Group
Sourced in United States, Germany
Embelin is a laboratory equipment product manufactured by Merck Group. It is a white to yellowish-brown crystalline powder that is used as a research tool in various scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Dmem f12, by Merck Group (2 mentions)
Quercetin, by Merck Group (2 mentions)
Ethanol, by Merck Group (1 mentions)
Oxacillin, by Merck Group (1 mentions)
Tetracycline, by Merck Group (1 mentions)
Methanol, by Lach-Ner (1 mentions)
Nucview 488 caspase 3 substrate, by Biotium (1 mentions)
Dmem high glucose, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Gemcitabine hydrochloride, by Merck Group (1 mentions)
Resazurin sodium salt, by Merck Group (1 mentions)
Geneprint 10 system, by Promega (1 mentions)
Carboplatin, by Enzo Life Sciences (1 mentions)
Strail apo2l, by Thermo Fisher Scientific (1 mentions)
Anacardic acid, by Merck Group (1 mentions)
Garcinol, by Enzo Life Sciences (1 mentions)
Fla 2000, by Fujifilm (1 mentions)
14c acetyl coa, by PerkinElmer (1 mentions)
Celltiter glo assay, by Promega (1 mentions)
18 protocols using embelin
1
Antibiotics and Embelin Combination
2
Drosophila Longevity Assay with Embelin
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Drosophila crosses were performed at 25 °C, 12 h light/dark cycle, on Formula 4–24 Instant Drosophila medium (Carolina) supplemented with 5% inactivated yeast. DMSO (100%) (Sigma-Aldrich) or embelin (Sigma-Aldrich) were added to the medium at a final DMSO concentration of 0.3% (42 mM). Drosophila behavioral assays and pupae formation were performed as described elsewhere.
3
Culturing Human Cell Lines for Experimental Analysis
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The human GCT cell line KGN (Riken BioResource Research Center, Ibaraki, Japan) was cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F12 (DMEM/F12, Sigma-Aldrich, St. Louis, MO, USA) with 5% fetal bovine serum (FBS) (Gibco, Waltham, MA, USA). Normal human fibroblast cells (F202 and N60, generously provided by Ted Tredget, University of Alberta) and normal human kidney cells (NKC, isolated from the proximal tubule, provided by Ron Moore) were cultured in DMEM/high glucose (Sigma-Aldrich) with 10% FBS. All culture media were supplemented with 2 mM L-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin) (Gibco). Cell line authentication was performed for KGN using short tandem repeat DNA profiling (Promega GenePrint 10 System, Madison, WI, USA) at the Genetic Analysis Facility at the Centre for Applied Genomics of The Hospital for Sick Children (Toronto, ON, Canada).
PAC-1 was generously provided by Paul Hergenrother (University of Illinois) and Hoechst 33,342 was generously provided by Linda Pilarski (University of Alberta). Other chemicals include recombinant human TRAIL (sTRAIL/Apo2L, Peprotech #310-04); carboplatin (Enzo Life Sciences #400-041); NucView 488 Caspase-3 substrate (Biotium #30029); and resazurin sodium salt (R7017), embelin (E1406), and gemcitabine hydrochloride (G6423-10) from Sigma-Aldrich.
PAC-1 was generously provided by Paul Hergenrother (University of Illinois) and Hoechst 33,342 was generously provided by Linda Pilarski (University of Alberta). Other chemicals include recombinant human TRAIL (sTRAIL/Apo2L, Peprotech #310-04); carboplatin (Enzo Life Sciences #400-041); NucView 488 Caspase-3 substrate (Biotium #30029); and resazurin sodium salt (R7017), embelin (E1406), and gemcitabine hydrochloride (G6423-10) from Sigma-Aldrich.
4
HAT Acetylation Activity Assay of NP Protein
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The procedures followed to perform this study were modified from a previous report (21 (link)). The recombinant proteins of NP (1 μg) were incubated with 1 μg of the recombinant protein of histone acetyltransferase (P300/CBP (Enzo Life Sciences), GCN5 or PCAF) and 7.4 kBq of [14C]acetyl-CoA (Perkin Elmer) at 30 °C for 2 h in buffer containing 50 mm Tris-HCl, pH 8.0, 10% glycerol, 1 mm DTT, and 10 mm sodium butyrate. To detect [14C] radioactivity, reactions were separated in 10% SDS-PAGE gels, after which an imaging plate was exposed to the gel for several days. Signals were detected using a fluoroimage analyzer (FLA-2000, Fujifilm). Anacardic acid (Sigma), garcinol (Enzo Life Sciences), and embelin (Sigma) prepared in DMSO were incubated with NP and a HAT for 30 min at 30 °C. The RNP was purified from virions of A/Puerto Rico/8/34 (PR8, H1N1) as described previously (51 (link)).
5
Compound Screening in T Lymphocytes
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Human peripheral blood T lymphocytes were treated for 24 h with the following compounds: Atropine (VWR International, Darmstadt, Germany), Artemisinin (MP Biomedicals, Eschwege, Germany), Embelin (Sigma-Aldrich), Rocaglamide, Camphor (MP Biomedicals), Caffeine (Sigma-Aldrich), Luteolin (Sigma-Aldrich), Curcumin (Alexis Biochemicals, San Diego, CA, USA), Wogonin (Wako Pure Chemical Industries, Osaka, Japan), β-Escin (MP Biomedicals), Berberine (Biomol, Hamburg, Germany), Triptolide (Enzo Life Sciences), Chrysin (Sigma-Aldrich), Baicalein (Biomol), Resveratrol (MP Biomedicals), Quercetin (Sigma-Aldrich), Emodin (Biotrend Chemikalien GmbH), Baicalin (Sigma-Aldrich) and Coumarin (Fisher Scientific, Schwerte, Germany). Cell viability was determined by the Cell-Titer Glo assay (Promega) according to the manufacturer's instructions.
6
Phytochemical Analysis and Cytotoxicity Evaluation
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Ethanol (95%, AR grade), acetonitrile (HPLC grade), and methanol (HPLC grade) were purchased from RCI Labscan, Bangkok, Thailand. Methanol (AR grade) was purchased from Honeywell, Charlotte, NC, USA. Deionized water was obtained using a water purification system from Thermo Scientific Co. (Waltham, MA, USA). Gallic acid, quercetin, and kaempferol were purchased from the TOKYO chemical industry Co., Ltd., Tokyo, Japan. Sodium carbonate, embelin, and doxorubicin hydrochloride were purchased from Sigma-Aldrich, St. Louis, MO, USA. Folin–Ciocalteu, orthophosphoric acid, and dimethylsulfoxide were obtained from Merck, Darmstadt, Germany. McCoy’s 5A medium, Eagle’s Minimum Essential Medium, and Dulbecco’s phosphate-buffered saline were purchased from Lonza, Walkersville, MD, USA. Penicillin, streptomycin, Dulbecco’s Modified Eagle Medium, fetal bovine serum, and 0.25% Trypsin-EDTA (Ethylenediaminetetraacetic acid) were purchased from Gibco, Waltham, MA, USA.
7
Screening Natural Antiviral Compounds
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Twelve natural antiviral compounds of plant origin were selected on the basis of their published in vitro or vivo antiviral activities. All cell culture grade natural compounds viz. rutin, quercetin, menisdaurin, β-sitosterol, hesperidin, psoralen, bergenin, azadirachtin, baccatin III, lupeol, embelin and naringenin were purchased (Sigma-Aldrich, Germany). The approved anti-HBV nucleoside analog, lamivudine (3TC; Sigma-Aldrich, Germany) served as standard (positive control).
8
Cholangiocarcinoma Cell Lines for Research
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Human malignant cholangiocarcinoma cell lines used in this study were KMCH [36] (link), Mz-ChA-1 [37] (link), and HuCCT cells [38] (link). The highly tumorigenic rat cholangiocarcinoma cell BDEneu was a kind gift from Alphonse Sirica (Virginia Commonwealth University) [39] (link). Human cells were grown in DMEM with high glucose supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 µg/ml), G418 (50 µg/ml), and insulin (0.5 µg/ml) at 37°C with 5% CO2 in a humidified chamber. BDEneu cells were grown in DMEM supplemented with 10% FBS, human transferrin (5 µg/ml), and insulin (0.5 µg/ml). Embelin was from Sigma-Aldrich and was resuspended in dimethylsulfoxide (DMSO). Staurospirine was from Fisher and was used at 1 µg/mL final concentration. Cells were treated with 0–50 µM Embelin for 2–48 hours, as indicated in the figure legends, and compared to DMSO-treated cells (vehicle). Recombinant human TRAIL was obtained from R&D Systems and used at a final concentration of 4–8 ng/mL.
9
Preparation of Chemical Stock Solutions
10
Embelin Modulation of Apoptotic Pathways
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Embelin was purchased from Sigma-Aldrich (St. Louis, MO). A 100 mM solution of Embelin was prepared in dimethyl sulfoxide, stored as small aliquots at -20°C and then diluted as needed in cell culture medium. A 8M solution of lithium chloride was obtained from Sigma-Aldrich and diluted as needed in cell culture medium. FBS and RPMI 1640 medium were purchased from HyClone (Logan, UT). The Neon transfection system, JC-1 assay kit and COX-4 antibody were from Life Technologies (Carlsbad, CA). The bicinchoninic acid (BCA) protein assay kit and ECL kit were from Thermo Scientific (Rockford, IL). Antibodies against phospho-Akt (Ser 473), GSK-3β, phospho-GSK-3β, or Bcl-2 were from Cell Signaling Technology (Beverly, MA). Antibodies against Total-Akt, VDAC1, Bcl-xL, Bax, β-catenin, cyclin D1, GAPDH or goat anti-rabbit IgG-Texas Red and Ultra Cruz mounting medium were from Santa Cruz Biotechnology (Santa Cruz, CA). Cyclooxygenase-2 (COX-2), Mcl-1, cytochrome c, AIF, β-catenin, c-myc or histone H1 antibodies were from Millipore Co. (Bedford, MA). All other chemicals were of the highest purity or molecular biology grade and were obtained from commercial sources.
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