We performed a high throughput screen of twelve chemotherapeutic drugs that are known ABCG2 substrates on the mouse G3 MB Myc1 neurosphere, as previously described (22 (link)). Briefly, 1000 cells per well were plated in 384-well plates. After 24 hr, half of the plates were treated with 10 µM of FTC (Alexis Biochemical, San Diego, CA, USA), 10 µM of Ko143 (Tocris Bioscience, Bristol, UK), or 10 µM of Tariquidar (Sigma-Aldrich, USA), incubated for 30 minutes, and all plates were treated with 28 nl of compound in a dose-response (final drug concentration 9.3 µM to 0.5 nM). After 72 hr, the cell number was determined in each well using CellTiter-Glo© (Promega). The luminescence signal was measured in an automated Envision plate reader (Perkin-Elmer). Luminescence data were normalized by log10-transformation prior to calculating the percentage of inhibition using the following equation: 100 × (negative control mean − compound value)/(negative control mean − positive control mean). Cells treated with 1% DMSO served as negative control with 0% inhibition. Cells treated with 35 µM cycloheximide were used as positive control with 100% inhibition.
Tariquidar
Manufactured by Merck Group
Sourced in United States
Tariquidar is a laboratory reagent manufactured by Merck Group. It is a P-glycoprotein inhibitor used in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Ko143, by Merck Group (4 mentions)
Verapamil, by Merck Group (3 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (2 mentions)
Acetonitrile, by J&K Scientific (2 mentions)
Paclitaxel, by Merck Group (2 mentions)
Doxorubicin, by Merck Group (2 mentions)
Celltiter glo, by Promega (1 mentions)
Envision plate reader, by PerkinElmer (1 mentions)
Ko143, by Bio-Techne (1 mentions)
Ix81 microscope, by Olympus (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Cell culture reagents, by Thermo Fisher Scientific (1 mentions)
Dp71 camera, by Olympus (1 mentions)
Fetal calf serum (fcs), by Natocor (1 mentions)
Sklb610, by Selleck Chemicals (1 mentions)
Prism 8, by GraphPad (1 mentions)
96 well plate, by Merck Group (1 mentions)
Ondansetron hydrochloride, by Merck Group (1 mentions)
N benzylbenzamide, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
15 protocols using tariquidar
1
Screening ABCG2 Inhibitors in Mouse G3 MB
2
Investigating P-Glycoprotein and HIF-1α Expression
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Cell culture reagents were obtained from Invitrogen Life Technologies (Carlsbad, United States). Fetal calf serum (FCS) was purchased from Natocor (Córdoba, Argentina). Antibodies were purchased from Santa Cruz (rabbit polyclonal anti-p65 cat# SC-8008 and polyclonal rabbit anti-Epo-R), Abcam (mouse polyclonal anti-P-gp, cat# ab3083), and Sigma (polyclonal rabbit antiHIF-1α cat# HPA001275). Poly-L-lysine, DAPI (4′6-diamidino-2-phenylindole), Rhodamine 123 (Rho123), 3rd generation P-gp inhibitor Tariquidar and other chemicals were obtained from Sigma (United States). Fluorescent secondary antibodies were purchased from Jackson Immunoresearch (United States). Microscopic images were taken using an Olympus IX- 81 microscope equipped with a DP71 camera (Olympus, Japan).
3
Cell Viability and Apoptosis Analysis
4
Growth Inhibition Assay for Cell Lines
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For growth-inhibition assays, cells were seeded into 96-well plates (Sigma-Aldrich) in 50 µL of normal culture medium. MRC-5 and eIMS cells were seeded at 2.0 × 103 cells/well and Karpas299 cells at 2.5 × 104 cells/well. Serial dilutions or single concentrations of BV (Slade Health Pty Ltd), MMAE (MedChem Express) and tariquidar (Sigma-Aldrich) were added to growth media and incubated under normal culture conditions of 5% O2 for 72 h prior to assessment by resazurin assay. Responses were analysed by four-parameter logistic model by non-linear regression in GraphPad Prism 8.1.2 (GraphPad Software, La Jolla, CA, USA) to calculate the GI50 value for each cell type.
5
Analytical Procedures for Drug Quantification
6
Cell Culture Reagents and Assays
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Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI-1640 medium, fetal calf serum (FCS), phosphate-buffered saline (PBS), trypsin-ethylenediaminetetraacetic acid (EDTA), l -glutamine, penicillin, and streptomycin were purchased from Gibco, Invitrogen (Carlsbad, CA, USA). An Annexin V:FITC Apoptosis Detection Kit was purchased from BD Pharmingen (San Diego, CA, USA). A Tools Cell Counting Kit (CCK-8) was purchased from Biotools Co., Ltd. (Taipei, Taiwan). Tariquidar, Ko143, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Citarinostat (ACY-241) was purchased from Selleckchem (Houston, TX, USA).
7
Evaluating Antiviral Compounds against SARS-CoV-2
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Dulbecco’s modified Eagle’s medium (DMEM) and phosphate-buffered saline (PBS) were purchased from Life Technologies (Carlsbad, CA, USA). Fetal bovine serum (FBS) was obtained from Eurobio. Remdesivir (GS-5734) and GC376 were purchased from Selleck Chemicals (Houston, TX, USA). Camostat mesylate, tariquidar, and dimethylsulfoxide (DMSO) were obtained from Sigma (Saint Louis, MO, USA). The lichen compounds were resuspended in DMSO at 100 mM.
8
Synthesis and Characterization of Thienorphine Derivatives
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Thienorphine hydrochloride (99.5% in purity, Figure 1 ) and thienorphine-3-glucuronide (99% in purity, Figure 1 ) were synthesized using the chemical synthesis laboratory of the Institute. Tariquidar, propranolol, verapamil, probenecid, quercetin, dexamethasone, methotrexate, and collagenase were purchased from Sigma (St. Louis, MO, USA). MDR1, MRP2 and BCRP membranes as well as an ATPase assay kit were purchased from BD Gentest Co. (Woburn, MA). Acetonitrile was of HPLC grade (J&K Chemical LTD), and other reagents were all of an analytical grade.
9
Fluorescein Annexin V Apoptosis Assay
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Fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit was purchased from BD Pharmingen (San Diego, CA, USA). Tools Cell Counting (CCK-8) kit was acquired from Biotools Co., Ltd. (Taipei, Taiwan). TP-3654 was obtained from Selleckchem (Houston, TX, USA). ABCB1 reference inhibitor tariquidar, ABCG2 reference inhibitor Ko143, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise.
10
Anticancer Drug Delivery Optimization
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TP (Fig. 1 ) was obtained from National Institutes for Food and Drug Control (Beijing, China) with the purity >99%. Tariquidar was purchased from Sigma (St. Louis, MO, USA). The siRNA was chemically synthesized by Ribobio Co., Ltd (Guangzhou, China). The rabbit anti-P-gp was obtained from Abcam (Cambridge, UK). The mouse anti-Bcl-2 and anti-Bax were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Pierce Biotechnology (Thermo Fisher Scientific, Rockford, IL, USA). Acetonitrile and Methanol were of HPLC grade (J&K Chemical LTD) and other reagents were all of analytical grade.
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