4 hydroxytamoxifen 4 oht

Manufactured by Merck Group

Sourced in United States, United Kingdom, Germany, Switzerland

4-hydroxytamoxifen (4-OHT) is a selective estrogen receptor modulator (SERM) commonly used as a laboratory reagent. It functions by binding to and modulating the activity of estrogen receptors. 4-OHT is utilized in various research applications to investigate cellular signaling pathways and mechanisms related to estrogen receptor regulation.

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Lab products found in correlation

Tamoxifen, by Merck Group (15 mentions) 4 oht, by Merck Group (14 mentions) 17β estradiol e2, by Merck Group (13 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (10 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions) Corn oil, by Merck Group (8 mentions) Etoposide, by Merck Group (8 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions) Puromycin, by Merck Group (6 mentions) Progesterone, by Merck Group (6 mentions) L glutamine, by Thermo Fisher Scientific (6 mentions) Penicillin, by Thermo Fisher Scientific (6 mentions) Streptomycin, by Thermo Fisher Scientific (6 mentions) Mcf 7, by American Type Culture Collection (5 mentions) Dimethyl sulfoxide (dmso), by Merck Group (5 mentions) Doxycycline, by Merck Group (5 mentions) Dexamethasone (dex), by Merck Group (5 mentions) Estradiol e2, by Merck Group (4 mentions) Ici 182 780, by Bio-Techne (4 mentions) Hepes, by Thermo Fisher Scientific (4 mentions)

282 protocols using 4 hydroxytamoxifen 4 oht

Cell Line Culture and Genetic Manipulation

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PMBL cell lines MedB-1, Karpas1106, and U2940, were cultured in RPMI 1640 medium supplemented with 10% FCS, glutamine, and antibiotics as described [23 (link)]. MedB-1 cells stably expressing a constitutively active form of human FOXO1 in-frame with the modified tamoxifen-specific version of the murine estrogen receptor-α ligand-binding domain (FOXO1-ER) were established by infection with pCFG5-FOXO1(A3)ER retroviral vector followed by selection with 100 μg of zeocin as we described earlier [11 (link)]. Control cells were transduced with empty pCFG5-IEGZ vector. FOXO1ER construct was induced by 4-hydroxytamoxifen (4-OHT) (Merck Millipore, Schwalbach, Germany) at a final concentration of 200 nM. The small-molecular weight MYC inhibitor 10058-F4 was obtained from Sigma-Aldrich (Steinheim, Germany). The JAK2 inhibitor TG 101348 was obtained from Axon Medhem (Groningen, The Netherlands); 5-aza-dC and TSA were purchased from Calbiochem (Darmstadt, Germany).

Xie L., Ritz O., Leithäuser F., Guan H., Färbinger J., Weitzer C.D., Gehringer F., Brüderlein S., Holzmann K., Vogel M.J., Möller P., Wirth T, & Ushmorov A. (2014). FOXO1 downregulation contributes to the oncogenic program of primary mediastinal B-cell lymphoma. Oncotarget, 5(14), 5392-5402.

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ER-Asi SI-Mediated Cell Survival Assay

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U2OS cells with ER-AsiSI (19 (link)) were kindly provided as a gift by Tanya Paull (University of Texas at Austin, Austin, TX). ER-AsiSI U2OS cells were grown in DMEM, which contained 10% FBS (GE Healthcare). Subsequently, 1 × 10⁵ cells were seeded into a 6-well plate, and 4-hydroxytamoxifen (4-OHT) (catalog [cat] no. H7904, Merck) was added to obtain a final treatment concentration of 300 nM. After 4 h of 4-OHT treatment, the media were refreshed, and the cells were incubated for 6 additional days. Cell survival was determined with either a colony-forming assay with methylene blue staining (cat no. M9140, Merck) or the CellTiter-Glo assay, performed according to the manufacturer’s protocol (G9241, Promega).

Kwon T., Ra J.S., Lee S., Baek I.J., Khim K.W., Lee E.A., Song E.K., Otarbayev D., Jung W., Park Y.H., Wie M., Bae J., Cheng H., Park J.H., Kim N., Seo Y., Yun S., Kim H.E., Moon H.E., Paek S.H., Park T.J., Park Y.U., Rhee H., Choi J.H., Cho S.W, & Myung K. (2022). Precision targeting tumor cells using cancer-specific InDel mutations with CRISPR-Cas9. Proceedings of the National Academy of Sciences of the United States of America, 119(9), e2103532119.

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Estrogen Receptor Signaling Pathway

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17β-estradiol (E2) was purchased from Sigma-Aldrich (St. Louis, MO). Recombinant hPRL (Lot AFP795) was obtained from Dr. A.F. Parlow (National Hormone and Pituitary Program, NIDDK, National Institutes of Health, Torrance, CA). Type-I rat tail collagen (#CB354249) was obtained from Fisher Scientific (Pittsburgh, PA). Inhibitors used for these studies were purchased as follows: 4-hydroxy-tamoxifen (4-OHT) (#579002) from EMD Millipore (Billerica, MA), SFK inhibitor, PP-1 (#EI275) from Biomol International, LP (Plymouth Meeting, PA), and JAK2 inhibitor, BMS-911543 (#CT-BMS91) from Chemietek (Indianapolis, IN). Type-I collagenase (#17100–017) was purchased from Invitrogen (Grand Island, NY). Antibodies used in these studies were as follows: PRLR-ECD (#35–9200) and pSRC Y418 (#44660G) from Invitrogen (Grand Island, NY); ERK1/2 (#9102) and cleaved caspase-3 (#9661) from Cell Signaling Technology (Danvers, MA); cSRC (sc-18) and EGFR (sc-03) from Santa Cruz Biotechnology (Santa Cruz, CA); ERα (#NCL-ER-6F211/2) from Novocastra (Newcastle, United Kingdom); pan-actin (#125-ACT) from Phosphosolutions (Aurora, CO); Ki-67 (#AB15580) from Abcam (Cambridge, MA). Multiwell non-tissue culture-treated plates were obtained from Corning Life Sciences (#08–772–49 and #08–772–51, Tewksbury, MA). All other reagents were obtained from Fisher Scientific or Sigma-Aldrich.

Barcus C.E., Holt E.C., Keely P.J., Eliceiri K.W, & Schuler L.A. (2015). Dense Collagen-I Matrices Enhance Pro-Tumorigenic Estrogen-Prolactin Crosstalk in MCF-7 and T47D Breast Cancer Cells. PLoS ONE, 10(1), e0116891.

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MCF-7 Cell Culture and Tamoxifen Induction

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The human BC cell line MCF-7 was bought from ATCC. MCF-7 cells were all maintained in Dulbecco's modified essential medium (DMEM), supplemented with 10% fetal bovine serum (FBS, Pricella, Wuhan, China), in a 5% CO 2 incubator. MCF-7-TAM was induced by 20 μM 4-hydroxytamoxifen (4-OHT, Calbiochem, Merck Millipore Billerica, MA, USA) incubation of MCF-7 for one month.

Mingting D.u.a.n., Yun R.e.n., Jiwen Z.h.a.n.g., Tingting Z.h.a.n.g., Yanhong W.a.n.g, & Hongyan J.i.a. (2023). High Expression of TIMM17B Is a Potential Diagnostic and Prognostic Marker of Breast Cancer. Cellular and molecular biology (Noisy-le-Grand, France), 69(3).

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Generating Conditional Knockout Mice

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Flt1^Loxp/Loxp mice were obtained from Dr. Gua-Hua Fong [20 (link)]. Cdh5^CreERT2 mice were obtained from Dr. Yoshiaki Kubota [27 (link)]. B6Ros.Cg-Dmd^mdx-5Cv/J (mdx; JAX stock #002379) [52 (link)], B6.Cg-Tg(CAG-cre/Esr1*)5Amc/J (CAG^CreERTM; JAX stock #004682) [19 (link)] and D2.129(Cg)-Gt(ROSA)26Sor^{tm4(ACTB-tdTomato-EGFP)Luo}/J (Rosa26R^mTmG; JAX stock #007576) [53 (link)] mice were obtained from Jackson Laboratory. All the mice used in this study were listed in S1 Table. Colonies for all the mice were established in the laboratory. Cre recombination was induced using tamoxifen (TMX) (T5648, MilliporeSigma) dosed as 75 mg/kg body weight x 3 time over one week at 3–4 weeks of age unless otherwise specified. We also injected 4-hydroxy tamoxifen (4-OHT) (H6278, MilliporeSigma) dosed as 25 mg/kg body weight. Control mice contained the wild-type (WT) CreER allele or were injected with the vehicle (corn oil or 10% ethanol).

Verma M., Shimizu-Motohashi Y., Asakura Y., Ennen J.P., Bosco J., Zhou Z., Fong G.H., Josiah S., Keefe D, & Asakura A. (2019). Inhibition of FLT1 ameliorates muscular dystrophy phenotype by increased vasculature in a mouse model of Duchenne muscular dystrophy. PLoS Genetics, 15(12), e1008468.

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Lentiviral Transduction and Tamoxifen Induction

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For each replicate, 3×10⁶ cells were spinfected in 5 wells of a 12-well plate in medium supplemented with 8μg/mL polybrene (Millipore Sigma; TR-1003-G) and spun at 2,000 rpm for 2 hours at 37°C. 2X the amount of virus determined by MOI was added per well. After spinfection, media was replaced (without polybrene) and incubated overnight. The next morning, 5 wells from each replicate were pooled and split onto two 10 cm plates per replicate. Media was replaced containing 2μg/mL puromycin (Thermofisher Scientific; A1113803) after 6 hours. Media was changed every 2 days and puromycin was removed after 4 days. 4×10⁶ cells were collected and snap frozen in an ethanol, dry-ice bath from each replicate as the day 0 timepoint. 4×10⁶ cells were plated per 15 cm plate for a total of 2 plates per replicate. One plate from each replicate was treated with 15nM 4-Hydroxytamoxifen (4-OHT) (Millipore Sigma; H7904). Cells were cultured for an additional 16 days, changing media and 4-OHT every 2 days, and splitting cells every 4 days, always at a minimum of 4×10⁶ cells per 15 cm plate. 4×10⁶ cells were harvested on day 8 and day 16 per condition for each replicate and snap frozen.

Einstein J.M., Perelis M., Chaim I.A., Meena J.K., Nussbacher J.K., Tankka A.T., Yee B.A., Li H., Madrigal A.A., Neill N.J., Shankar A., Tyagi S., Westbrook T.F, & Yeo G.W. (2021). Inhibition of YTHDF2 triggers proteotoxic cell death in MYC-driven breast cancer. Molecular cell, 81(15), 3048-3064.e9.

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Synthesis and Dissolution of GSK Compounds

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GSK5182 was synthesized at Kyungpook National University (Daegu, Republic of Korea) and DGMIF (Daegu-Gyeongbuk Medical Innovation Foundation, Daegu, Republic of Korea), as described previously [10 (link),34 (link),43 (link),44 (link)], and dissolved in 30% polyethylene glycol 400 (PEG400, USB, Cleveland, OH, USA) or DMSO (MilliporeSigma, St. Louis, MO, USA). GSK4716 was purchased from Abcam (Cambridge, UK). 4-hydroxytamoxifen (4-OHT) and bisphenol A (BPA) were purchased from MilliporeSigma (St. Louis, MO, USA). XCT790 was purchased from TOCRIS (Bristol, UK)

Na S.Y., Kim K.S., Jung Y.S., Kim D.K., Kim J., Cho S.J., Lee I.K., Chung J., Kim J.S, & Choi H.S. (2022). An Inverse Agonist GSK5182 Increases Protein Stability of the Orphan Nuclear Receptor ERRγ via Inhibition of Ubiquitination. International Journal of Molecular Sciences, 24(1), 96.

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Expanding and Characterizing Mouse Tregs

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CD4⁺CD25⁺ Treg cells were purified from mouse lymph nodes and spleen according to the protocol provided by the manufacturer (Invitrogen, catalog no. 11463D). Treg cells were expanded according to the Treg Expansion Kit protocol (Miltenyi Biotec, catalog no. 130–095–925). Treg cells were treated with 0.5 μM 4-Hydroxytamoxifen (4-OHT) (Millipore Sigma, catalog no. H7904) for 3 days, 10 μM TMG (CarboSynth), or 100 μg/ml cycloheximide (CHX) (Millipore Sigma, catalog no. C7698) as indicated. To determine the purity, Treg cells were assessed by flow cytometry and immunoblot analysis.

Liu B., Salgado O.C., Singh S., Hippen K.L., Maynard J.C., Burlingame A.L., Ball L.E., Blazar B.R., Farrar M.A., Hogquist K.A, & Ruan H.B. (2019). The lineage stability and suppressive program of regulatory T cells require protein O-GlcNAcylation. Nature Communications, 10, 354.

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Timed Csf1r-Cre Lineage Tracing

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Male Csf1r-Cre^ER;TdTomato^f/f mice were crossed with TdTomato^f/f or Tdtomato^f/+ females to generate control TdTomato^f/f or Tdtomato^f/+ and experimental Csf1r-Cre^ER;TdTomato^f/f or Csf1r-Cre^ER;TdTomato^f/+ animals within the same litters. We did not see any spontaneous recombination (e.g. tdTomato fluorescence or RFP immunoreactivity) in control animals. Pregnancy was determined by the presence of a copulation plug (gestational day 0.5 (gd0.5)), and maternal weight was measured at gd0.5 and gd8.5 (to confirm pregnancy weight gain). Pregnant females were injected intraperitoneally (i.p.) with 10 mg/kg 4-hydroxytamoxifen (4-OHT, Millipore-Sigma cat #H6278) dissolved in corn oil (Sigma) at gd8.5 and euthanized at gd17.5 with CO₂ followed by rapid decapitation. Sex was recorded and is clearly noted throughout the manuscript.

Batorsky R., Ceasrine A.M., Shook L.L., Kislal S., Bordt E.A., Devlin B.A., Perlis R.H., Slonim D.K., Bilbo S.D, & Edlow A.G. (2023). Hofbauer cells and fetal brain microglia share transcriptional profiles and responses to maternal diet-induced obesity. bioRxiv.

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Cell Line Authentication and Treatment

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All cell lines were obtained from ATCC (American Type Culture Collection) and authenticated using short tandem repeat (STR) analysis. Cells were cultured in MEM or RPMI-1640 medium (MSKCC Media Prep, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA) and maintained at 37 °C and 5% CO₂ in humidified atmosphere. Reagents used for cell culture and treatments: fulvestrant (Selleckchem, USA), SNX-2112 (Selleckchem, USA), charcoal-stripped fetal bovine serum (CSS, Gibco, USA), 17β-estradiol (E2, Sigma-Aldrich, USA), 4-Hydroxytamoxifen (4-OHT, Sigma-Aldrich, USA).

Strillacci A., Sansone P., Rajasekhar V.K., Turkekul M., Boyko V., Meng F., Houck-Loomis B., Brown D., Berger M.F., Hendrickson R.C., Chang Q., de Stanchina E., Pareja F., Reis-Filho J.S., Rajappachetty R.S., Del Priore I., Liu B., Cai Y., Penson A., Mastroleo C., Berishaj M., Borsetti F., Spisni E., Lyden D., Chandarlapaty S, & Bromberg J. (2022). ERα-LBD, an isoform of estrogen receptor alpha, promotes breast cancer proliferation and endocrine resistance. NPJ Breast Cancer, 8, 96.

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