Human breast cancer cell line MCF-7 was obtained from American Type Culture Collection (Manassas, VA, USA). The HEK293 cell line was obtained from Clontech (Palo Alto, CA, USA). Short tandem repeat-based authentication of the cell line was verified by BEX Co., Ltd. (Tokyo, Japan). Long-term estrogen-deprived (LTED) cells were established through the long-term (>4 months) culturing MCF-7 cells without estrogen [47 (link)]. MCF-7 cells and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Nacalai Tesque, Kyoto, Japan) at 37 °C with 5% CO2. LTED cells and estrogen-treated cells were cultured in phenol red-free DMEM containing 5% charcoal-stripped FBS and 1% penicillin-streptomycin at 37 °C with 5% CO2. DMEM was purchased from Nacalai Tesque. Estradiol (E2) was purchased from Sigma-Aldrich.
Estradiol e2
Manufactured by Merck Group
Sourced in United States
Estradiol (E2) is a laboratory reagent used for various research and analytical applications. It is a naturally occurring steroid hormone that plays a crucial role in the female reproductive system. Estradiol is commonly used in biomedical research, pharmaceutical development, and clinical diagnostics to measure and analyze hormone levels.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Medroxyprogesterone acetate mpa, by Merck Group (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
4 hydroxytamoxifen 4 oht, by Merck Group (4 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
Tamoxifen, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (3 mentions)
Phenol red free dmem, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Gemini Bio (2 mentions)
4 hydroxytamoxifen, by Merck Group (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Opti mem medium, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (2 mentions)
Dextran coated charcoal, by Merck Group (2 mentions)
Ici 182 780, by Bio-Techne (2 mentions)
Docetaxel, by Merck Group (2 mentions)
Gefitinib, by Selleck Chemicals (2 mentions)
50 protocols using estradiol e2
1
Culturing Breast Cancer and HEK293 Cells
2
Estrogen-Deprived Breast Cancer Cell Lines
3
Immortalization of Myeloid Progenitors
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Immortalization of myeloid hematopoietic progenitors was performed by transduction of CD117 (c-Kit) positive bone marrow cells with a retrovirus expressing HoxB8 fused to the estrogen receptor21 (link). Cells were grown in 10 ng/ml IL-3 (Goldbio) and 1 μM estradiol (E2) (Sigma Aldrich). AML cell lines were obtained from ATCC between 2009–2014. They were validated by STR profiling in 2019 and undergo mycoplasma testing every 6 months. Myeloid progenitors, Nomo1, and U937 cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum (Gibco, 10438026), 1% penicillin/streptomycin (Gibco, 15140122), and 1% GlutaMAX (Gibco, 35050061). 293T packaging cells were grown in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% GlutaMAX. Proliferation was measured at the indicated times starting with a concentration of 10,000 cells/ml in the case of the immortalized murine myeloid progenitors and with 100,000 cells/ml for human cell lines. Cell number was determined by a hemocytometer counting chamber.
4
Antibody Validation Protocol
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Estradiol (E2) was purchased from Sigma-Aldrich. Antibodies against the following epitopes were used: Myc (9E10) and HA (12CA5) from Roche Applied Science (Germany), GFP (G1544) from Santa Cruz Biotechnology (USA), and α-Tubulin (B-5-1-2) from Sigma-Aldrich (USA).
5
Estrogen Receptor Activation Assay
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Estradiol (E2) was purchased from Sigma (USA). Premarin® tablets (0.625 mg) was purchased from Pfizer Inc. (NY, USA). MCF-7 cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher, USA), supplemented with 10% fetal calf serum (Biological Industries, SC, USA) and 100 μg/ml penicillin/streptomycin (Thermo Fisher, Yokohama, Japan), as described previously 10 (link). For estrogen-free experiments, the cells were seeded in phenol-red free DMEM (Thermo Fisher), and supplemented with 10% charcoal-dextran-treated fetal calf serum (Thermo Fisher) prior to treatment with the extracts. MCF-7 cells were transfected with 3XERE-TATA-luc and tk-Rluc, followed by incubation with the vehicle control, 10−10 M to 10-8 M E2, 1/1000 to 1/1 Premarin, 10-8 M to 10-6 M glabridin, or 50% ethanol extract licorice-based products. To minimize the effect on cells, 0.5 μL of the extract was added in 0.5 mL of the culture media (1/1000). A luciferase reporter vector containing and estrogen responsive element was transiently transfected into the estrogen receptor (ER)-alpha positive MCF-7 cells. DNA transfection and luciferase assay were performed as described previously 10 (link).
6
Mitochondrial Dynamics Visualization Assay
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Dulbecco’s modified eagle medium (DMEM), trypsin, fetal bovine serum (FBS), Lipofectamin 2000, and MitoTracker Red CMXRos (CMX) were obtained from Thermo Fisher Scientific (Thermo Fisher Scientific GmbH, Basel, Switzerland). Molecular weight markers, complete protease cocktail inhibitors, and Western blotting luminol reagent were purchased from Thermo Fisher Scientific, Roche (Mannheim, Germany) and Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), respectively. Geneticin (G418) was provided by Calbiochem (Merck, Kenilworth, NJ, USA). All other chemicals, including Hoechst 33342, staurosporine, estradiol (E2), and H2O2, were purchased from Sigma-Aldrich (St. Louis, MO, USA).
7
Estrogen Receptor Signaling Assay
8
MCF-7 Cell Culture and Validation
9
Estrogen Receptor Ligand Evaluation
10
Comparative Evaluation of Selective ER Modulators
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Fulvestrant (CAS No: 129453–61-8, > 99% purity) was purchased from MedChem Express. Estradiol (E2) (E8875), lasofoxifene (SML1026), 4-hydroxytamoxifen (H7904), and tamoxifen (T5648) were purchased from Sigma. Raloxifene (2280) was purchased from Tocris. GDC-0810 (S7855), bazedoxifene (S2128), and AZD9496 (S8372) were purchased from Selleckchem. GW5638 (5638), GW7604 (7604), and RU 58,668 (RU) were provided by Donald McDonnell (Duke University). G1T48 was provided by G1 Therapeutics, Inc., as analytical grade compound.
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