Ko143

Manufactured by Merck Group

Sourced in United States, France, Germany

Ko143 is a laboratory equipment product designed for scientific research and analysis. It serves as a tool for performing specific tasks or experiments within a controlled laboratory environment. The core function of Ko143 is to facilitate data collection and analysis, though its intended use may vary depending on the specific research or application requirements.

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Lab products found in correlation

88 protocols using ko143

Hoechst 33342 Staining for Side Population

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Forty-eight hours after transfection with specific siRNAs, CNE2 and 5-8F cells were trypsinized and resuspended at density of 1 × 10⁶ cells per ml. The DNA-binding dye, Hoechst 33342 (Sigma-Aldrich), was then added at a final concentration of 7.5 μg/ml and incubated for 90 min in the dark with periodic mixing. After washing twice with PBS, the cells were placed at 4 °C in the dark before flow cytometry (EPICS ALTRA Flow Cytosorter, Beckman Coulter, Indianapolis, IN, USA) using dual-wavelength analysis. However, a fraction of the cell preparation was incubated with 5 μM Ko143 (Ko143, a specific inhibitor of ABCG2, Sigma-Aldrich) for 10 min at 37 °C before adding Hoechst 33342 to determine whether Ko143 would block the fluorescent efflux of SP cell.

Liu Z.H., Hu J.L., Liang J.Z., Zhou A.J., Li M.Z., Yan S.M., Zhang X., Gao S., Chen L., Zhong Q, & Zeng M.S. (2015). Far upstream element-binding protein 1 is a prognostic biomarker and promotes nasopharyngeal carcinoma progression. Cell Death & Disease, 6(10), e1920-.

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Ruxolitinib Sensitivity in ABCG2-Expressing Cells

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K562 WT-, mock-, and ABCG2-transfected cells were treated with 0–200 µM ruxolitinib for 72 h to calculate the IC₅₀ of ruxolitinib. To treat the K562 WT-, mock-, and ABCG2-transfected cells, 30 µM ruxolitinib (IC₅₀ K562-WT), a potent and selective ABCG2 inhibitor at the concentration used, was used with or without 0.5 µM KO143 pre-treatment for 1 h (Sigma-Aldrich, St. Louis, MO, USA) [43 (link),44 (link)]. MPN and ABCG2_null RBCs were treated with 50 µM ruxolitinib for 72 h with or without 0.01 µM KO143 pre-treatment for 1 h. Cultured erythroid cells were treated with 0.1 µM ruxolitinib for 48 h with or without 1 µM KO143 pre-treatment for 1 h. DMSO (Sigma-Aldrich, St. Louis, MO, USA) was used as the solvent control. Cell viability was measured by Cell Counting Kit-8 (CCK8) (Sigma-Aldrich, St. Louis, MO, USA) and PS exposure was measured using an annexin V kit.

Buks R., Brusson M., Cochet S., Galochkina T., Cassinat B., Nemazanyy I., Peyrard T., Kiladjian J.J., de Brevern A.G., Azouzi S, & El Nemer W. (2021). ABCG2 Is Overexpressed on Red Blood Cells in Ph-Negative Myeloproliferative Neoplasms and Potentiates Ruxolitinib-Induced Apoptosis. International Journal of Molecular Sciences, 22(7), 3530.

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Measuring ABCG2 Expression in Cell Lines

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We determined the cell surface expression of ABCG2 in the stable HeLa cell lines and in transiently transfected HEK293 cells 48 h after transfection. Antibody labeling was performed in trypsinized cells with the ABCG2-specific 5D3 mouse monoclonal antibody (gift of Bryan Sorrentino, Division of Experimental Hematology, Department of Hematology/Oncology, St. Jude Children’s Research Hospital). Ko143 is an ABCG2 inhibitor that has an impact on ABCG2 conformation, thus helping 5D3 antibody recognition. 1 µM Ko143 (Sigma-Aldrich, cat. K2144) was added to the samples before the measurements. Alexa Fluor 647-labeled IgG2b (Thermo Fisher, cat. A-21242) was used as a secondary antibody. Propidium iodide (final concentration: 1.6 µg/ml, Sigma Aldrich, cat. 4170) was used for dead cell separation. Measurements were carried out in FACS Canto II after gating for live, EGFP positive cells.

Zámbó B., Mózner O., Bartos Z., Török G., Várady G., Telbisz Á., Homolya L., Orbán T.I, & Sarkadi B. (2019). Cellular expression and function of naturally occurring variants of the human ABCG2 multidrug transporter. Cellular and Molecular Life Sciences, 77(2), 365-378.

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Radiolabeled Compounds for Cellular Assays

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[¹⁴C]Urate (55.0 mCi/mmol), [³H]guanine (10.7 Ci/mmol), [³H]hypoxanthine (27.0 Ci/mmol), [³H]thymine (65.0 Ci/mmol), and [³H]uracil (42.8 Ci/mmol) were obtained from Moravek Biochemicals (Brea, CA), [¹⁴C]inulin (1.9 mCi/g) was from American Radiolabeled Chemicals (St. Louis, MO), and [³H]polyethylene glycol 4000 (PEG 4000, 1.5 mCi/g) was from PerkinElmer Life Sciences (Boston, MA). Unlabeled urate, guanine, hypoxanthine, thymine, and uracil were obtained from Wako Pure Chemical Industries (Osaka, Japan), and Ko143 was from Sigma‐Aldrich (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Wako Pure Chemical Industries and Invitrogen (Carlsbad, CA), respectively. Mouse monoclonal antibodies for the tag peptides of DYKDDDDK (FLAG) and hemagglutinin (HA) were obtained from Wako Pure Chemical Industries (product numbers of 014‐21881 and 018‐22381, respectively, for the anti‐FLAG and anti‐HA antibodies), and a mouse monoclonal antibody for β‐actin (product number A5441) and horseradish peroxidase‐conjugated goat anti‐mouse IgG (product number A8924) were from Sigma‐Aldrich. All other reagents were of analytical grade and commercially obtained.

Yasujima T., Murata C., Mimura Y., Murata T., Ohkubo M., Ohta K., Inoue K, & Yuasa H. (2018). Urate transport function of rat sodium‐dependent nucleobase transporter 1. Physiological Reports, 6(10), e13714.

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Characterization of Uric Acid Transporters

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The following compounds were purchased commercially from the sources indicated: allopurinol, benzbromarone, cyclosporine, D-fructose, elacridar, furosemide, hydrochlorothiazide, nicotinic acid, oxypurinol, rosuvastatin calcium salt, salicylic acid, 4-hydroxy chalcone (Wako Fine Chemical, Osaka, Japan); atorvastatin, chlorothiazide, febuxostat, mizoribine, pyrazinecarboxylic acid, ribavirin, tacrolimus, xylitol (Tokyo Chemical Industry, Tokyo, Japan); ethambutol, losartan (LKT Laboratories, St Paul, MN, USA); fenofibrate, probenecid, sulfasalazine, Ko143, ATP, AMP, creatine phosphate disodium salt tetrahydrate, creatine phosphokinase type I from rabbit muscle (Sigma-Aldrich, St. Louis, MO, USA); pyrazinamide (ACROS ORGANICS, Geel, Belgium); theophylline (Nacalai Tesque, Kyoto, Japan); and topiroxostat (MedChem Express, Princeton, NJ, USA). The [8-¹⁴C]-uric acid (53 mCi/mmol) was from American Radiolabeled Chemicals (St. Louis, MO, USA). All other chemicals used were commercially available and of analytical grade.

Miyata H., Takada T., Toyoda Y., Matsuo H., Ichida K, & Suzuki H. (2016). Identification of Febuxostat as a New Strong ABCG2 Inhibitor: Potential Applications and Risks in Clinical Situations. Frontiers in Pharmacology, 7, 518.

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Identification of Cancer Stem Cells

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An APC-conjugated anti-CD44 antibody (BD Bioscience, clone G44-26), a PE-Cy7-conjugated anti-CD24 antibody (BioLegend, clone LM5), an APC-conjugated anti-ABCG2 antibody (clone 5D3, BD Bioscience) and propidium iodide (5 μg ml⁻¹; BD Bioscience) were used for fluorescence-activated cell sorting analyses. To detect the SP fraction, MCF7 and ZR75-1 cells were stained with 5 μg ml⁻¹ Hoechst 33342 (Invitrogen) in the presence or absence of 1 μM Ko143 (Sigma) at 37 °C for 90 min. Flow cytometric analysis and cell sorting were performed using a JSAN cell sorter (Bay bioscience) and the results were analysed with FlowJo software.

Takahashi R.U., Miyazaki H., Takeshita F., Yamamoto Y., Minoura K., Ono M., Kodaira M., Tamura K., Mori M, & Ochiya T. (2015). Loss of microRNA-27b contributes to breast cancer stem cell generation by activating ENPP1. Nature Communications, 6, 7318.

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Assessing BCRP Inhibition on SN-38 Cytotoxicity

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Cells were seeded at 10,000 cells/well in 96-well plates and allowed to adhere for 48 h at 37 °C. Cell lines were exposed to SN-38 with or without Ko143, a specific BCRP inhibitor (Sigma Aldrich) for 72 h. Cell viability was assessed using MTT assays with triplicate determinations from three independent biological passages.

Jandu H., Aluzaite K., Fogh L., Thrane S.W., Noer J.B., Proszek J., Do K.N., Hansen S.N., Damsgaard B., Nielsen S.L., Stougaard M., Knudsen B.R., Moreira J., Hamerlik P., Gajjar M., Smid M., Martens J., Foekens J., Pommier Y., Brünner N., Schrohl A.S, & Stenvang J. (2016). Molecular characterization of irinotecan (SN-38) resistant human breast cancer cell lines. BMC Cancer, 16, 34.

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Quantitative Analysis of Lipophilic Drug Transport

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Dimethyl sulfoxide (DMSO), insulin, 1,6-diphenyl-1,3,5-hexatriene (DPH), stearic acid, oleic acid, tamoxifen, valspodar (PSC833), calcein-am, sulforhodamine 101, Ko143, MK571, and mitoxantrone were purchased from Sigma-Aldrich (St Louis, MO, USA). Hydrocortisone hemisuccinate was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Penicillin/streptomycin, fetal bovine serum (FBS) were purchased from Biochrom (Berlin, Germany). 7-β-NBD-taurocholate was custom-synthesized as described by Schneider et al. [34 (link)] PPC and PI were obtained from Lipoid (Ludwigshafen am Rhein, Germany). EPL (Essentiale Forte 300 mg) was obtained from Sanofi. All other chemicals were purchased from commercial sources and were of the highest purity available.

Wupperfeld D., Fricker G., Bois De Fer B., Frank L., Wehrle A, & Popovic B. (2022). Essential phospholipids decrease apoptosis and increase membrane transport in human hepatocyte cell lines. Lipids in Health and Disease, 21, 91.

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Biotin and 4-PBA Compound Treatment

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Biotin (Sigma-Aldrich, cat. B4501) and 4-PBA (Sigma-Aldrich, cat. P21005) were dissolved in distilled water and used at 100 μM and 1 mM final concentrations, respectively. MG132 (Sigma-Aldrich, cat. M7449), Bafilomycin A1 (BAF) (Sigma-Aldrich, cat. B1793), and Ko143 (Sigma-Aldrich, cat. K2144) were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, cat. 276855) and applied at 2 μM, 10 nM, and 1 μM final concentrations, respectively. For solvent controls, distilled water and DMSO were used accordingly.

Bartos Z, & Homolya L. (2021). Identification of Specific Trafficking Defects of Naturally Occurring Variants of the Human ABCG2 Transporter. Frontiers in Cell and Developmental Biology, 9, 615729.

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Oxysterol Regulation of LXR Signaling

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All stocks were stored at −20°C. scOHCs were from Avanti (Alabama, US) and stored as 10 mM stocks in nitrogen flushed ethanol: 24OHC (#700061), 25OHC (#700019), 26OHC (#700021). Epirubicin (Cayman, UK Cat:12091) was stored at 10 mM in nuclease free water and protected from light. GSK2033 (ToCris, Abindon, UK – #5694) at 20 mM diluted in ETOH. ABC inhibitors: MK-571 (Cambridge Bioscience – Cat: 10029–1mg-CAY) and KO143 (Sigma – Cat: K2144–1mg) were diluted in DMSO, while Verapamil (Insight Biotechnology – Cat: sc-3590) was diluted in NFW; all at 10 mM. TaqMan assays (Thermo Fisher, Paisley, #4331182): LXRα [Hs00172885_m1] LXRβ [Hs01027215_g1], Pgp [Hs00184500_m1], ABCA1 [Hs01059137_m1], HPRT1 [Hs02800695_m1 ]. Origine trisilencer complexes (Maryland, US): LXRα #SR322981, LXRβ #SR305039). Antibodies: Pgp (Santa Cruz Biotech, CA, US - #sc73354), CYP27A1 (Abcam, Cambridge, UK - #ab126785), CYP46A1 (Abcam, Cambridge, UK - #ab198889), CH25H (Bioss, MA, US - #bs6480R), LXRα (R&D System, Minneapolis, US - #PP-PPZ0412–00), LXRβ (Active Motif, Carslbad, US - #61177). Antibody validation is described in detail in Supplementary Information.

Hutchinson S.A., Websdale A., Cioccoloni G., Røberg-Larsen H., Lianto P., Kim B., Rose A., Soteriou C., Pramanik A., Wastall L.M., Williams B.J., Henn M.A., Chen J.J., Ma L., Moore J.B., Nelson E., Hughes T.A, & Thorne J.L. (2021). Liver x receptor alpha drives chemoresistance in response to side-chain hydroxycholesterols in triple negative breast cancer. Oncogene, 40(16), 2872-2883.

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