Cells were grown for 4 days in a hormone-stripped DMEM devoid of phenol red and supplemented with 3% steroid-depleted, charcoal-treated foetal calf serum (Biowest, Riverside, MO, USA); 104 cells per well were plated onto a 96-well plate in charcoal-stripped medium. The following day, the cells were treated with 4-hydroxy-tamoxifen (OH-Tam, Sigma) at different concentrations for 72 h. Cell viability was analysed using the Cell Titer 96® aqueous one solution Cell Proliferation kit from (Promega, Fitchburg, WI, USA). Briefly, the reagent (MTS) was added in each well, incubated for 1 h at 37 °C, and the absorbance was recorded at 490 nm following the manufacturer’s recommendations.
4 hydroxy tamoxifen oh tam
Manufactured by Merck Group
Sourced in United States
4-hydroxy-tamoxifen (OH-Tam) is a metabolite of the drug tamoxifen. It is used as a laboratory reagent for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Foetal calf serum, by Biowest (1 mentions)
Celltiter 96 aqueous one solution cell proliferation kit, by Promega (1 mentions)
17β estradiol e2, by Merck Group (1 mentions)
Hotstarttaq plus dna polymerase, by Qiagen (1 mentions)
L trans epoxysuccinyl leucylamido 4 guanidino butane e 64, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Charcoal stripped fbs, by Thermo Fisher Scientific (1 mentions)
Ctso plasmid, by OriGene (1 mentions)
Olaparib, by Selleck Chemicals (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Progesterone, by Merck Group (1 mentions)
Oh tam, by Merck Group (1 mentions)
C57bl 6 mice, by Janvier Labs (1 mentions)
4 androstenedione ad, by Merck Group (1 mentions)
4 protocols using 4 hydroxy tamoxifen oh tam
1
Cell Viability Assay with Tamoxifen
2
Gene Expression Regulation in Breast Cancer
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Dulbecco's minimum essential medium (DMEM), glutamine and penicillin/streptomycin/glutamine stock mix were purchased from Life Technologies, Inc. (Carlsbad, CA, USA). Fetal bovine serum (FBS) and charcoal-stripped FBS were from Invitrogen (Carlsbad, CA, USA). L-trans-Epoxysuccinyl-leucylamido (4-guanidino) butane (E-64) was from Sigma-Aldrich (St. Louis, MO USA). CTSO, MTDH, PABPC4L, LMNA, EEFiA1and control small interfering RNAs (siRNA) were purchased from Dharmacon (Thermo Scientific Dharmacon, Inc.). CTSO plasmid was purchased from OriGene (Rockville, MD, USA). Affinity purified rabbit and mouse antibodies against human BRCA1 and CTSO were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). ZNF423 antibody was purchased from Abcam (Cambridge, MA, USA). Actin, MTDH, PABPC4L, LMNA, and EEFiA1 antibodies were from cell signaling (Danvers, MA, USA). For standard PCR, HotStart Taq Plus DNA Polymerase was used (Qiagen, Germantown, MD, USA). Reagents and primers for real time PCR were purchased from Qiagen (Valencia, CA, USA). The protease inhibitor cocktail kit was obtained from Pierce Biotechnology (Rockford, IL, USA). 17β-estradiol (E2) and 4-hydroxytamoxifen (OH-TAM) were purchased from Sigma Aldrich (Saint Louis, MO USA). Olaparib was from Selleckchem (Houston, TX, USA).
3
Multicolor Lineage Tracing in Mice
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C57Bl6 mice were purchased from Janvier Labs. Cx3cr1EGFP/+ (Jung et al., 2000 (link)), Csf1r-Gal4VP16/UAS-ECFP (MacBlue; Ovchinnikov et al., 2008 (link)), and Ccr2−/− mice were intercrossed to generate MacBlue × Cx3cr1EGFP/+, MacBlue × Cx3cr1EGFP/+ × Ccr2−/− litermate mouse strains. These strains, and MMTV PyMT-P2A-mCherry-P2A-OVA (PyMT-ChOVA) mice (Engelhardt et al., 2012 (link)) were bred at Pitié-Salpêtrière animal facility. Csf1rMeriCreMer; Rosa26tdTomato-LSL (Qian et al., 2011 (link)) and Tnfrsf11aCre; Rosa26YFP-LSL (Maeda et al., 2012 (link)) were bred at the Memorial Sloan Kettering Research Animal Resource Center. For the labeling of the EMP lineage, pulse labeling was performed in Csf1rMeriCreMer; Rosa26tdTomato-LSL E8.5 embryos with 4-hydroxytamoxifen (OH-TAM, Sigma-Aldrich). Embryonic development was estimated considering the day of vaginal plug formation as 0.5 d after coitum. Cre recombination was induced by a single injection of 37.5 mg per kg (body weight) of OH-TAM into pregnant females. OH-TAM was supplemented with 18.75 mg per kg (body weight) progesterone (Sigma-Aldrich) to counteract the mixed estrogen agonist effects of tamoxifen, which can result in fetal abortions.
All mice were maintained under SPF conditions and used between 8 and 14 wk old except for PyMT-ChOVA that develop primary breast tumors and lung metastases at around 25 wk.
All mice were maintained under SPF conditions and used between 8 and 14 wk old except for PyMT-ChOVA that develop primary breast tumors and lung metastases at around 25 wk.
4
Establishing Endocrine-Resistant Breast Cancer Cell Lines
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From the ERα MCF-7-derived breast cancer cell line stably transfected with the human aromatase gene (MCF-7 control) as previously reported [37 (link)], different cell lines were established, namely: a tamoxifen-resistant cell line (Res-Tam), a fulvestrant-resistant cell line (Res-Fulv), an anastrozole-resistant cell line (Res-Ana) [38 (link)], and a letrozole-resistant cell line (Res-Let) [39 (link)]. The Res-Tam cell line was established by exposing the MCF-7 control cells during 25 weeks to increasing concentrations (1 and 3 μM) of 4-hydroxy-tamoxifen (OH-Tam, Sigma, St Louis, MO, USA) in Dulbecco’s Modified Eagle Medium without phenol red, supplemented with 3% steroid depleted, dextran-coated and charcoal-treated fetal calf serum (DCC medium) containing 25 nM 4-androstenedione (AD) (Sigma, Saint Louis, MO, USA). All of the human cell lines were maintained at 37 °C in the appropriate medium supplemented with 10% foetal calf serum.
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