Z 4 hydroxytamoxifen

Manufactured by Merck Group

Sourced in United States, Germany

(Z)-4-hydroxytamoxifen is a chemical compound that serves as a laboratory reagent. It functions as a selective estrogen receptor modulator (SERM), a class of compounds that can act as either estrogen agonists or antagonists depending on the tissue type and biological context.

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Lab products found in correlation

Tamoxifen, by Merck Group (7 mentions) Penicillin, by Thermo Fisher Scientific (3 mentions) Streptomycin, by Thermo Fisher Scientific (3 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions) 17β estradiol, by Merck Group (2 mentions) Rpmi 1640 glutamax medium, by Thermo Fisher Scientific (2 mentions) Rmil 7, by Thermo Fisher Scientific (2 mentions) Fetal calf serum (fcs), by Harvard Bioscience (2 mentions) β mercaptoethanol, by Thermo Fisher Scientific (2 mentions) Antibiotic antimycotic solution, by Merck Group (2 mentions) Dasatinib, by Selleck Chemicals (2 mentions) Pegfr y1068 d7a5, by Cell Signaling Technology (2 mentions) Her2 erbb2 d8f12, by Cell Signaling Technology (2 mentions) Per s118α 16 j4, by Cell Signaling Technology (2 mentions) C6orf97 antibody, by GeneTex (2 mentions) V5 ma5 15253 antibody, by Thermo Fisher Scientific (2 mentions) Pher2 y1221 1222, by Cell Signaling Technology (2 mentions) Bez235, by Selleck Chemicals (2 mentions) Integrin β1 d2e5, by Cell Signaling Technology (2 mentions) Pher2 y877, by Cell Signaling Technology (2 mentions)

47 protocols using z 4 hydroxytamoxifen

Conditional Knock-out Neurons in vitro

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Neurons from cortex and hippocampus were isolated from postnatal mice at p1–5 as previously described66 (link). Cells were transfected at 7–9 days in vitro (DIV) using an optimized calcium phosphate protocol66 (link).
To initiate homologous recombination in neurons from floxed animals expressing a tamoxifen-inducible Cre recombinase cultured neurons were treated with 0.25 μM (Z)-4-hydroxytamoxifen (Sigma) immediately after plating (DIV0). Equal concentrations of tamoxifen (0.25 μM) were used during medium renewal on DIV1 and DIV2. Ethanol was added to control neurons in an amount equal to the tamoxifen concentration (0.25 μM). In some cases, homologous recombination was induced at a later stage by treating cultured neurons from floxed animals expressing a tamoxifen-inducible Cre recombinase with 0.25 μM (Z)-4-hydroxytamoxifen (Sigma) at DIV8. For rescue experiments homologous recombination was induced by applying the tamoxifen at DIV0. AP-2μ-mRFP construct was introduced at DIV8 and the neurons were analysed at DIV14.

Kononenko N.L., Claßen G.A., Kuijpers M., Puchkov D., Maritzen T., Tempes A., Malik A.R., Skalecka A., Bera S., Jaworski J, & Haucke V. (2017). Retrograde transport of TrkB-containing autophagosomes via the adaptor AP-2 mediates neuronal complexity and prevents neurodegeneration. Nature Communications, 8, 14819.

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Isolation and Culture of Developing Mouse Cochleae

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Inner ears were isolated at E17.5, P1, P3 or P6 animals as described previously (Tao and Segil, 2015 (link)). Cochleae were then micro-dissected under a stereo microscope in a laminar flow hood in ice-cold Ca²⁺- and Mg²⁺-free DPBS. After removal of lateral wall and spiral ganglion tissues, cochleae were cultured on SPI black filter membranes (SPI Supplies) in L15 Leibovitz media (Hyclone) supplemented with N2 (Invitrogen, 1:100), B27 (Gibco, 1:50), Penicillin G (Sigma, 100U/ml), EGF(Shenandoah Biotechnology, 5ng/ml), FGF (Shenandoah Biotechnology, 2.5ng/ml) and HEPES (Invitrogen, 10µM). To lineage-trace supporting cells in Lfng-CreERT2/tdTomato animals, (Z)-4-Hydroxytamoxifen (Sigma) was included in the culture medium at a concentration of 0.02mg/ml for the first 24 hours. The following inhibitors were used at the indicated concentrations: 10µM DAPT (Calbiochem), 10µM GSK-343 (courtesy gift from GlaxoSmithKline PLC), 200nM TSA (Sigma), 20μM GSK-LSD1 (Sigma) or an equivalent amount of DMSO (VWR).

Tao L., Yu H.V., Llamas J., Trecek T., Wang X., Stojanova Z., Groves A.K, & Segil N. (2021). Enhancer decommissioning imposes an epigenetic barrier to sensory hair cell regeneration. Developmental cell, 56(17), 2471-2485.e5.

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Regulation of Breast Cancer Cell Lines

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Parental and ERβ1-expressing MCF7 cells [12 (link)] and doxycycline-inducible Hs578T-ERβ1 cells [8 (link)] were cultured as previously described. Doxycycline-inducible ERβ1-expressing MDA-MB-231 cell lines were established using the T-REx™ System (Invitrogen) as previously described [9 (link)] and were maintained in DMEM/F12 medium supplemented with 10% FBS, 1% AA, 5 mg/L blasticidin S and 500 mg/L zeocin. Charcoal-stripped fetal bovine serum (CS-FBS) was purchased from Gemini Bio-Products (West Sacramento, CA). 17β-estradiol (E2), (Z)-tamoxifen, (Z)-4-hydroxy-tamoxifen and doxycycline (Dox) were purchased from Sigma-Aldrich (St. Louis, MO). (Z)-endoxifen was synthesized by the National Cancer Institute (Bethesda, MD). The ERβ-specific agonists; DPN, WAY200070, FERb 033 and Liquiritigenin, as well as the pure ER antagonist ICI 182,780, were purchased from Tocris Bioscience (Bristol, United Kingdom).

Reese J.M., Suman V.J., Subramaniam M., Wu X., Negron V., Gingery A., Pitel K.S., Shah S.S., Cunliffe H.E., McCullough A.E., Pockaj B.A., Couch F.J., Olson J.E., Reynolds C., Lingle W.L., Spelsberg T.C., Goetz M.P., Ingle J.N, & Hawse J.R. (2014). ERβ1: characterization, prognosis, and evaluation of treatment strategies in ERα-positive and -negative breast cancer. BMC Cancer, 14, 749.

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Astrocyte Culture and Ca2+ Imaging

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Primary cortical astrocytes were cultured according to standard protocols, modified from (Schildge et al., 2013 ). Briefly, cortices of P4–7 GLAST-CreER;mGCaMP3/+;IP3R2−/−triple transgenic mouse pups were dissociated using a Neural Tissue Dissociation Kit (P) (Miltenyi Biotec). Isolated cells were plated on a T-75 flask coated with poly-l-lysine (Sigma-Aldrich) and fed with DMEM (Life Technologies) supplemented with 10 % heat-inactivated FBS (Life Technologies) and 1 % penicillin-streptomycin (Life Technologies). After 7 days, astrocytes formed a confluent layer at the bottom of the flask. These cells were then plated on 12 mm cover glass (Thermo Fisher Scientific) coated with poly-l-lysine (Sigma-Aldrich) and Natural Mouse Laminin (Thermo Fisher Scientific) at a density of 20,000 cells per cover glass. One day after plating, expression of mGCaMP3 was induced by applying 1 μM (Z)-4-hydroxytamoxifen (H7904, Sigma-Aldrich). At least 14 days later, astrocyte Ca²⁺ transients were imaged using a Zeiss LSM 710 microscope, as described below. To block mPTP activity, astrocyte cultures were incubated for 1 hour prior to Ca²⁺ imaging in mPTP inhibitors [Cyclosporin A (inhibits cyclophilin D, Tocris, 20 μM; takes long time to dissolve and needs filtration of remaining precipitates) and Rotenone (Mitochondrial complex I inhibitor, Tocris, 10 μM)].

Agarwal A., Wu P.H., Hughes E.G., Fukaya M., Tischfield M.A., Langseth A.J., Wirtz D, & Bergles D.E. (2017). Transient opening of the mitochondrial permeability transition pore induces microdomain calcium transients in astrocyte processes. Neuron, 93(3), 587-605.e7.

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Skeletal Muscle Dissociation and FACS Isolation of FAPs

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Details of skeletal muscle dissection have been previously described (13 (link)). In brief, muscle was dissected from Acvr1^{[R206H]FlEx/+}; Gt(ROSA26)Sor^CreERT2/+ mice and dissociated using the Skeletal Muscle Dissociation Kit (Miltenyi Biotec) and gentleMACS Octo Dissociator with heaters (Miltenyi Biotec), in accordance with the manufacturer’s instructions. Following centrifugation at 300g and 4°C for 10 minutes, the supernatant was discarded, and the pellet was resuspended in growth media (Dulbecco’s modified Eagle medium (DMEM; Life Technologies) with 50 U/mL penicillin, 50 μg/mL streptomycin (Gibco), and 16.6% fetal bovine serum (FBS; lot 192K18, Avantor). Cells were then plated onto tissue culture flasks (Corning). FACS was performed on single cells incubated with anti–mouse PDGFRA APC (clone APA5, eBioscience) to label FAPs, as previously described (13 (link), 35 (link)). FACS-isolated FAPs were seeded at a density of 2000 cells/cm² onto tissue culture flasks (Corning) in growth media and maintained at 37°C in a humidified atmosphere with 5% CO₂. Media were changed every other day. Prior to use, FAPs were treated with 2 μM (Z)-4-hydroxytamoxifen (Sigma-Aldrich) for 48 hours to induce inversion of the R206H-containing exon. All experiments were conducted with FAPs passaged fewer than 3 times.

Aykul S., Huang L., Wang L., Das N.M., Reisman S., Ray Y., Zhang Q., Rothman N., Nannuru K.C., Kamat V., Brydges S., Troncone L., Johnsen L., Yu P.B., Fazio S., Lees-Shepard J., Schutz K., Murphy A.J., Economides A.N., Idone V, & Hatsell S.J. (2022). Anti-ACVR1 antibodies exacerbate heterotopic ossification in fibrodysplasia ossificans progressiva (FOP) by activating FOP-mutant ACVR1. The Journal of Clinical Investigation, 132(12), e153792.

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Mouse Models of Soft Tissue Sarcoma

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All animal experiments were performed according to protocols approved by the Duke University Institutional Animal Care and Use Committee. Mouse models of soft tissue sarcoma were generated in a mixed 129/SVJae and C57BL/6 background using a combination of previously described alleles: Pax7^Cre-ER-T2 (provided by Chen-Ming Fan, Carnegie Institution for Science) [20 (link)], p53^FL (provided by Anton Berns, The Netherlands Cancer Institute) [44 (link)], Hif1a^FL (provided by Randall Johnson, University of Cambridge) [45 (link)], and Nras^LSL-G12D [46 (link)] and ROSA26^mTmG [47 (link)] which were obtained from The Jackson Laboratory. (Z)-4-hydroxytamoxifen (Sigma Aldrich), which is referred to as 4-OHT, was first dissolved in 100% ethanol at a concentration of 250mg/ml, and then diluted with corn oil (Sigma Aldrich) to a final working solution concentration of 25mg/ml. Primary mouse soft tissue sarcomas were generated in the mouse hind limb by intramuscular (IM) injection of 20μl 4-OHT working solution via a 27.5 gauge insulin syringe (Terumo).

Zhang M., Qiu Q., Li Z., Sachdeva M., Min H., Cardona D.M., DeLaney T.F., Han T., Ma Y., Luo L., Ilkayeva O.R., Lui K., Nichols A.G., Newgard C.B., Kastan M.B., Rathmell J.C., Dewhirst M.W, & Kirsch D.G. (2015). HIF-1α regulates the response of primary sarcomas to radiation therapy through a cell autonomous mechanism. Radiation research, 183(6), 594-609.

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Inducible Runx1 Deletion in Mice

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Homozygous male ROSA^mT/mG mice were mated with heterozygous female Runx1^MerCreMer mice overnight (at 18:00). Female mice were examined for post-coital plug the following morning (at 08:00), and those with a plug were considered pregnant and timed E0.5. Pregnant mice received a single dose of 0.1 mg/g body weight (Z)-4-Hydroxytamoxifen supplemented with 0.05 mg/g body weight progesterone resuspended in corn oil (all from Sigma-Aldrich) via intraperitoneal (i.p.) injection at E8. progesterone supplementation counteracts estrogen receptor antagonism by tamoxifen to circumvent fetal abortions.

Eddins D.J., Kosters A., Waters J., Sosa J., Phillips M., Yadava K., Herzenberg L.A., Kuipers H.F, & Ghosn E.E. (2021). Hematopoietic stem cell requirement for macrophage regeneration is tissue-specific. Journal of immunology (Baltimore, Md. : 1950), 207(12), 3028-3037.

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Fibroblast response to fenofibrate and tamoxifen

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Patient fibroblasts (WESP) were maintained in minimum essential medium α (MEM α) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO, USA). Adult normal human dermal fibroblasts (NHDF) were acquired from PromoCell and grown in Fibroblast Growth Medium 2 with SupplementMix (PromoCell GmbH, Heidelberg, Germany). Fibroblasts were grown at 37 °C and 5% CO₂.
Cells were treated with 1, 10, or 25 µM fenofibrate (Sigma, St. Louis, MO, USA) or 0.1, 0.5, or 1 µM (Z)-4-hydroxytamoxifen (Sigma-Aldrich, St. Louis, MO, USA) for 24 h prior to cell pellet collection for downstream analyses. For combination treatments, 25 µM fenofibrate and 0.5 µM (Z)-4-hydroxytamoxifen were used to treat the cells for 24 h prior to cell pellet collection.

Galano M., Ezzat S, & Papadopoulos V. (2022). SCP2 variant is associated with alterations in lipid metabolism, brainstem neurodegeneration, and testicular defects. Human Genomics, 16, 32.

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Inducible Transgene Expression in T Cells

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T cell numbers were adjusted to 5 × 10⁴ transduced T cells/mL and transgene transcription was induced by the addition of the indicated concentration of (Z)-4-hydroxytamoxifen (Sigma-Aldrich, St. Louis, MO, USA), (Z)-endoxifen (Axon Medchem, Groningen, The Netherlands) or a 1:9 mixture thereof (stock: 1 µM (Z)-4-hydroxytamoxifen, 9 µM (Z)-endoxifen) directly to the culture medium. Transgene expression was analyzed at the indicated time point by flow cytometry.

Kotter B., Engert F., Krueger W., Roy A., Rawashdeh W.A., Cordes N., Drees B., Webster B., Werchau N., Lock D., Dapa S., Schneider D., Ludwig S., Rossig C., Assenmacher M., Mittelstaet J, & Kaiser A.D. (2021). Titratable Pharmacological Regulation of CAR T Cells Using Zinc Finger-Based Transcription Factors. Cancers, 13(19), 4741.

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Preparation of Pharmaceutical Compounds

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Sunitinib for in vitro studies was obtained from Sigma-Aldrich (St Louis, MO) and dissolved in dimethyl sulfoxide to a final concentration of 500 nM. Sunitinib for mouse gavage was obtained from Selleck Chemicals (Munich, Germany) and dissolved in olive oil. Vandetanib was obtained from AstraZeneca Pharmaceuticals (Macclesfield, United Kingdom) and dissolved in dimethyl sulfoxide to a final concentration of 10 μM. z-4-Hydroxytamoxifen was obtained from Sigma-Aldrich and dissolved in ethanol to a final concentration of 10 μM. Glial cell line–derived neurotrophic factor (GDNF) was purchased from Sigma-Aldrich and dissolved in phosphate-buffered saline and to a final concentration of 2.5 ng/mL.

Spanheimer P.M., Cyr A.R., Gillum M.P., Woodfield G.W., Askeland R.W, & Weigel R.J. (2014). Distinct Pathways Regulated by RET and Estrogen Receptor in Luminal Breast Cancer Demonstrate the Biological Basis for Combination Therapy. Annals of surgery, 259(4), 793-799.

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