Cells were placed in steroid hormone depleted media (phenol red free DMEM, 10% six-times charcoal/dextran stripped fetal bovine serum, 2% L-glutamine, and 1% penicillin/streptomycin) for 3 days. Cells were plated in 6 well plates, transfected with glucocorticoid/progesterone response element Luciferase (0.75 μg) and CMV-β-galactosidase (0.25 μg) reporter plasmids [38 (link), 39 (link)] using Lipofectamine 3000 (Life Technologies). For transient expression experiments, T47D PR KO cells were co-transfected with 100 ng LHLCA expression plasmid containing either wild type PR-A or PR-B, or Y890C PR-A or PR-B along with the reporter plasmids. After 5 h, medium was changed. The next day, cells were treated with either ethanol (EtOH) or 10 nM R5020 (promegestone; Perkin Elmer) for 24 h. Luciferase (Promega) and β-galactosidase activity (Tropix) were assayed following manufacturer protocols.
R5020
Manufactured by PerkinElmer
Sourced in United States
The R5020 is a compact and versatile laboratory instrument designed for precise data collection and analysis. It features a high-resolution data acquisition system and advanced signal processing capabilities. The core function of the R5020 is to provide accurate and reliable measurements for a variety of scientific applications.
Automatically generated - may contain errors
Lab products found in correlation
Ru486, by Merck Group (3 mentions)
Mycoalert mycoplasma detection kit, by Lonza (3 mentions)
Dexamethasone 21 mesylate dex 21 mesylate dm, by Steraloids (2 mentions)
Dual luciferase reporter assay, by Promega (2 mentions)
R5020, by Merck Group (2 mentions)
Progesterone p4, by Merck Group (2 mentions)
17β estradiol e2, by Merck Group (2 mentions)
Charcoal stripped serum, by Biowest (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Dmem f12, by Roche (1 mentions)
S1250, by Selleck Chemicals (1 mentions)
Nod cg prkdcscid il2rgtm1wjl szj mice, by Charles River Laboratories (1 mentions)
Balb c, by Jackson ImmunoResearch (1 mentions)
C57bl 6, by Jackson ImmunoResearch (1 mentions)
Collagenase a, by Roche (1 mentions)
Mycoplasma infection, by Lonza (1 mentions)
16 protocols using r5020
1
Transcriptional Regulation by Progesterone Receptors
2
Quantifying Kiss Production in mHypoA-51 Neurons
3
Dexamethasone Derivatives Assay
4
Dexamethasone Derivatives Assay
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Dexamethasone (Dex), 11-deoxycorticosterone [DOC], progesterone (Prog), and RU486 are from Sigma (St. Louis, MO), R5020 from PerkinElmer (Boston, MA), and dexamethasone 21-mesylate (Dex-21-mesylate; DM) from Steraloids (Newport, RI). Dexamethasone oxetanone [Dex-Ox] was prepared as described [30 ]. Dual-luciferase reporter assay is from Promega (Madison, WI).
5
Breast Cancer Cell Line Maintenance and Hormone Treatments
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The breast cancer cell line T47D was obtained from the University of Colorado Cancer Center Tissue Culture core and was maintained in minimal Eagle’s medium, 5% fetal bovine serum (FBS), 1× non-essential amino acids, 1 × 10−9 M insulin, 0.1 mg/mL sodium pyruvate, and 2 mM L-glutamine. Generation of breast cancer cell lines UCD4 and UCD65 has been previously described [20 (link)]. The UCD4 and UCD65 cell lines were maintained in DMEM/F-12 1:1 with 10% FBS, 1 × 10−9 M cholera toxin, and 1 × 10−9 M insulin. Cell lines were authenticated using short tandem repeat (STR) analysis using the University of Arizona Genetics Core (University of Arizona, Tucson, AZ, USA). All cell lines were routinely tested for mycoplasma contamination using the MycoAlert mycoplasma detection kit (Lonza, Basel, Switzerland). In vitro hormone experiments were performed using phenol red-free media plus charcoal stripped FBS with the same additives described above. Hormone treatment was used as follows: vehicle (0.2% ethanol), 17-β-estradiol (E2), 10−8 M (Sigma-Aldrich, St. Louis, MO, USA); R5020, 10−8 M (PerkinElmer, Waltham, MA, USA); or progesterone (P4), 10−7 M (Sigma-Aldrich), or the combination of E2 plus R5020 (both 10−8 M) for 24 h unless otherwise indicated.
6
Isolation and Culture of Primary Mouse Mammary Organoids
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Animal experiments were performed in accordance with protocol approved by the Service de la Consommation et des Affaires vétérinaires of Canton de Vaud (VD 1865.3, VD 1865.4). NOD. Cg‐Prkdcscid Il2rgtm1Wjl/SzJ mice (Charles River), BalbC, and C57Bl6 breeders were purchased from Jackson Laboratories. Mammary glands were isolated from 18‐ to 35‐week‐old mice, lymph nodes removed, minced with surgical blades, and digested in DMEM/F12, 1% penicillin/streptomycin B with collagenase A 0.25 mg/ml (Roche). Tissue was concomitantly stimulated with hormones for 1, 2, or 6 h and centrifuged at 652 g for 5 min. Fat was removed, and pellet containing organoids washed with phosphate‐buffered saline (PBS), resuspended 5 min in 3 ml red cell blood lysis buffer and washed twice with PBS. Hormones and drug used: LNG, Sigma L0551000‐30MG; GSN, Sigma SML0292, DSG, Sigma SML0356‐5MG; DSP, Sigma SML0147‐10MG; CMA, Sigma C5145‐1G; CPA, Sigma C3283000‐30MG, R5020, Perkin Elmer NLP004005, bicalutamide, Toronto research laboratory B382000, enzalutamide, Selleck Chemicals S1250.
7
Breast and Kidney Cell Culture Protocol
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T47D were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS), 2% penicillin-streptomycin (Life Technologies), and insulin (10 μg/ml). Cos7 cells were maintained in DMEM, supplemented with 10% FBS and 2% penicillin-streptomycin (Life Technologies). All cell lines were grown in a humidified atmosphere with 5% CO2 at 37 °C, authenticated by Eurofins and tested for Mycoplasma infection (Lonza, Rockland, ME, USA).
Prior to experiments, when it was indicated, cells were grown in phenol red-free medium supplemented with 10% charcoal-stripped serum (Biowest). Cells were then treated with 10 nM of R5020 (Perkin Elmer) or E2 (Sigma) for the indicated times.
Prior to experiments, when it was indicated, cells were grown in phenol red-free medium supplemented with 10% charcoal-stripped serum (Biowest). Cells were then treated with 10 nM of R5020 (Perkin Elmer) or E2 (Sigma) for the indicated times.
8
Progestin Receptor Signaling Assay
9
Breast Cancer Cell Line Maintenance and Hormone Treatments
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The breast cancer cell line T47D was obtained from the University of Colorado Cancer Center Tissue Culture core and was maintained in minimal Eagle's medium, 5% fetal bovine serum (FBS), 1X non-essential amino acids, 1x10-9 M insulin, 0.1 mg/mL sodium pyruvate, and 2 mM L-glutamine.
Generation of breast cancer cell lines UCD4 and UCD65 has been previously described [20] (link). The UCD4 and UCD65 cell lines were maintained in DMEM/F-12 1:1 with 10% FBS, 1x10-9 M cholera toxin, and 1x10-9 M insulin. Cell lines were authenticated using short tandem repeat (STR) analysis using the University of Arizona Genetics Core (University of Arizona, Tucson, AZ). All cell lines were routinely tested for mycoplasma contamination using the MycoAlert mycoplasma detection kit (Lonza, Basel, Switzerland). In vitro hormone experiments were performed using phenol red-free media plus charcoal stripped FBS with the same additives described above. Hormone treatment was used as follows: vehicle (0.2% ethanol), 17--estradiol (E2), 10-8 M (Sigma-Aldrich, St. Louis, MO); R5020, 10-8 M (PerkinElmer, Waltham, MA); or progesterone (P4), 10-7 M (Sigma-Aldrich), or the combination of E2 plus R5020 (both 10-8 M) for 24 h unless otherwise indicated.
Generation of breast cancer cell lines UCD4 and UCD65 has been previously described [20] (link). The UCD4 and UCD65 cell lines were maintained in DMEM/F-12 1:1 with 10% FBS, 1x10-9 M cholera toxin, and 1x10-9 M insulin. Cell lines were authenticated using short tandem repeat (STR) analysis using the University of Arizona Genetics Core (University of Arizona, Tucson, AZ). All cell lines were routinely tested for mycoplasma contamination using the MycoAlert mycoplasma detection kit (Lonza, Basel, Switzerland). In vitro hormone experiments were performed using phenol red-free media plus charcoal stripped FBS with the same additives described above. Hormone treatment was used as follows: vehicle (0.2% ethanol), 17--estradiol (E2), 10-8 M (Sigma-Aldrich, St. Louis, MO); R5020, 10-8 M (PerkinElmer, Waltham, MA); or progesterone (P4), 10-7 M (Sigma-Aldrich), or the combination of E2 plus R5020 (both 10-8 M) for 24 h unless otherwise indicated.
10
Screening of Selective Estrogen Receptor Modulators
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17β-estradiol (E2), and Tam, and 4OH-Tam were obtained from Sigma (St. Louis, MO), and R5020 was purchased from PerkinElmer Life Sciences (Waltham, MA) These agents were dissolved in DMSO (Sigma) and added to culture medium at the time of assays. MTT dye ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was obtained from Sigma. The compounds screened in bioassays were obtained from the NCI/DTP Open Chemical Repository through the DTP website: http://dtp.cancer.gov in powder form.
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