MCF-10A and MCF-12A cells were cultured in Matrigel following an adapted protocol from Debnath et al.24 (link). Briefly, 8-well chamber permanox slide (Thermo Scientific) was coated with Growth Factor Reduced Matrigel Matrix, (Matrigel), (BD Biosciences). 5000 MCF-12A cells were seeded in DMEM/F12 phenol red free media supplemented with 2% charcoal stripped horse serum (Invitrogen), 5 ng/mL EGF (Invitrogen), 0.5 μg/mL hydrocortisone, 100 ng/mL cholera toxin, 10 μg/mL insulin (Roche), 1X pen/strep (Wisent) and 2% Matrigel. MCF-12A cells were treated with BPA or BPS (0.1, 1, and 10 μM) or 1 nM oestrogen (E2) (Sigma) or 0.1% ethanol. The treatment media was changed every 4 days and the cells were grown until time points at days 8, 16 and 25. For the experiments using ICI 182,780 (Sigma), the cells were plated in Matrigel as described above in the presence or absence of 1 µM ICI 182,780.
Ici 182 780
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany
ICI 182,780 is a synthetic steroidal estrogen receptor antagonist. It functions as a selective estrogen receptor downregulator (SERD) by binding to and inhibiting the estrogen receptor. This product is intended for use in research and laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Tamoxifen, by Merck Group (10 mentions)
17β estradiol e2, by Merck Group (9 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
17β estradiol, by Merck Group (6 mentions)
Estradiol, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
4 hydroxytamoxifen, by Merck Group (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Hydrocortisone, by Merck Group (3 mentions)
Genistein, by Merck Group (3 mentions)
Insulin, by Merck Group (3 mentions)
Fulvestrant, by Merck Group (3 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (3 mentions)
Insulin, by Roche (2 mentions)
Resveratrol, by Merck Group (2 mentions)
Trypsin, by Merck Group (2 mentions)
β estradiol e2, by Merck Group (2 mentions)
Tnf α, by R&D Systems (2 mentions)
Activated charcoal, by Merck Group (2 mentions)
Sb203580, by Selleck Chemicals (2 mentions)
73 protocols using ici 182 780
1
Culturing MCF-10A and MCF-12A Cells in Matrigel
2
Estrogen Receptor Signaling Inhibition
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Cells were treated with 10 nmol/L E2 in phenol red‐free DMEM supplemented with 2% charcoal‐stripped fetal bovine serum. Control cells were exposed to the same volume of medium without E2. For the inhibition experiments performed using ICI 182780 (Sigma), sirtinol (Sigma), and Compound C (Sigma), new DMEM supplemented with 2% charcoal‐stripped fetal bovine serum was added and maintained for 30 minutes, 1, 3, 6, and 24 hours before the addition of the inhibitors: ICI 182780 (1 μmol/L), sirtinol (50 μmol/L), Compound C (10 μmol/L), or G15 (2 μmol/L; Cayman).
3
Manipulation of Progesterone Receptor Signaling
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T47D-Y (PR-null) and MCF7 (PR-low) (19 (link)) cells were stably transfected with pSG5 or pSG5-PR-B and pSV-Neo or pIRES or pIRES-PR-B using FuGENE reagent (ThermoFisher) to generate multiple vector matched clones. Cells were cultured as previously described (12 (link), 19 (link)). Additional MCF7 cells were obtained from ATCC (PR-A/B+), cultured as indicated, and stably infected with retrovirus expressing LXSN, LXSN-wtPELP1, or LXSN-ΔNLS PELP1 (34 (link)). MCF7C4-12 (PR-null), MCF7L (PR-A/B+), BT474 (PR-A/B+), and MCF7 1GX (PR-A/B+) cells were cultured as described (12 (link), 26 (link), 36 (link), 50 (link)), MCF7L PR knockdown cells were generated using lentivirus shRNAs targeting PR (5 sequences) or GFP in pLKO (ThermoFisher).
Cells were treated with estradiol (1nM), tamoxifen (100nM), and ICI 182,780 (1uM) obtained from Sigma-Aldrich (St. Louis, MO); LY-294002 (10uM), U0126 (10uM), and PP2 (10uM) obtained from Calbiochem (Darmstadt, Germany); R5020 (10nM) purchased from NEN Life Science Products (Boston, MA); and IGF1 (5nM) from GroPep Bioreagents (Thebarton SA, Aus).
Cells were treated with estradiol (1nM), tamoxifen (100nM), and ICI 182,780 (1uM) obtained from Sigma-Aldrich (St. Louis, MO); LY-294002 (10uM), U0126 (10uM), and PP2 (10uM) obtained from Calbiochem (Darmstadt, Germany); R5020 (10nM) purchased from NEN Life Science Products (Boston, MA); and IGF1 (5nM) from GroPep Bioreagents (Thebarton SA, Aus).
4
Immunoblot Analysis of Heregulin-β1 Signaling
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Treatments consisted vehicle, heregulin-β1 (HRG-β1, R&D systems), E2, 4-hydroxy-tamoxifen (OHT), or ICI-182,780 (Sigma-Aldrich). The immunoblotting has been previously described [17 (link)] and details of antibodies are provided in Additional file 1 . Although the majority of immunoblots were reprobed with antibodies against ACTB (β-actin) as a loading control, only representative data per batch of cell lysates are shown.
5
Pharmacological Inhibition Assay
6
Chondrocyte Isolation and Culture
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We used the following reagents: DMEM/F12 (Gibco, USA), fetal bovine serum (FBS) (BI, Israel), trypsin (Sigma, USA), collagenase type II (Sigma, USA), D-Hanks and PBS (Solarbio, Beijing, China), Annexin V-FITC/PI kit (BD, USA), primary antibody of anti-α1 (Proteintech, Wuhan, China), E2 (Sigma, USA), collagen II (Sigma, USA), ICI182780 (Sigma, United Kingdom), and secondary antibody (goat anti-rabbit) (Proteintech, Wuhan, China).
7
Estrogen Receptor Pathway Modulation
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C3G was purchased from Polyphenols AS Laboratories (Hanabryggene Technology Center, Norway). 17β-Estradiol (E2) and the ERs antagonist ICI 182,780, Dimethylsulfoxide (DMSO), phosphate buffered saline (PBS), and other chemicals were purchased from Sigma-Aldrich (Sigma, USA); ERα agonist PPT and ERβ agonist DPN were from Tocris Biosciences (Tocris Biosciences, UK).
8
Microglia Cell-Based Inflammation Study
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BV-2 cells (EOC-20 CRL-2469; ATCC), immortalized microglia cell line derived from the brain of an apparently normal 10 day old mouse, were cultured in DMEM/F12 medium with 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 95% air and 5% CO2. Cells were plated into 24-well plates at the density of 5 × 105/well. Cells were divided into four groups: control group (Ctr group), lipopolysaccharide (LPS, L4391, Sigma-Aldrich, St. Louis, MO, United States) group (LPS group), LPS+TNA group (LPS+TNA group), and LPS+TNA+ICI group (LPS+TNA+ICI group). Cells were treated in absence or presence of LPS (1 μg/mL) for overnight. For LPS+TNA group and LPS+TNA+ICI group, cells were treated with concentrations of TNA at 10μM for 24 h. Cells in LPS+TNA+ICI group were treated with ICI 182, 780 (Sigma-Aldrich, St. Louis, MO, United States) at 5 μmol/l for 24h. At last cells were collected for quantitative RT-PCR and Western blotting.
IL-10 knockdown was conducted by 300 nM IL-10 siRNA plasmids (sc-39,635, Santa Cruz Biotechnology, Inc.) or a nonsense scramble siRNA (sc-37,007, Santa Cruz Biotechnology, Inc.) transfection in BV-2 cells. After transfection for 24 h, cells were treated with LPS+TNA for another 24 h. Then cells were collected for analyzing.
IL-10 knockdown was conducted by 300 nM IL-10 siRNA plasmids (sc-39,635, Santa Cruz Biotechnology, Inc.) or a nonsense scramble siRNA (sc-37,007, Santa Cruz Biotechnology, Inc.) transfection in BV-2 cells. After transfection for 24 h, cells were treated with LPS+TNA for another 24 h. Then cells were collected for analyzing.
9
Breast Cancer Cell Line Culturing and Estrogen Treatment
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MCF7, T47D and MDA-MB231 cell lines purchased from NCCS (Pune, India) were maintained as previously described.66 (link) MCF10A, a kind gift from Dr Annapoorni Rangarajan (IISc, Bangalore, India) was maintained as previously described.67 (link) The cells maintained for 48–72 h in phenol-free medium prior to hormone treatments were then treated with estrogen (Sigma, St Louis, MO, USA) at a concentration of 100 nM for 24–48 h. During inhibition studies, MCF7 cells were treated with 10 μM of tamoxifen and 1 μM of ICI182,780 (Sigma) for 6 h prior to hormone treatment. To study the direct effect of estradiol in CASP7 expression, MCF7 cells were treated with 10 μg/ml of cyclohexamide and 5 μM of actinomycin D (Sigma) for 30 min followed by hormone treatment for 24–48 h.
10
Estrogenic Receptor Agonists and Hep G2 Cells
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β-Estradiol (052-04041; Wako Pure Chemical Industries, Ltd, Osaka, Japan), 4-hydroxytamoxifen (H7904; Sigma-Aldrich, St Louis, MO, USA), and ICI182780 (ICI, I4409; Sigma-Aldrich) were purchased. The E2 enzyme immunoassay kit (No 582251) was purchased from Funakoshi (Tokyo, Japan). EDTA (15111-45), NaCl (31320-34), and Tris (35406-91) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). ERα-specific agonist 4,4′, 4′-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT, CAS No: 263717-53-9) was purchased from Sigma-Aldrich (H6036). ERβ-specific agonist 2,3-bis(4-hydroxyphenyl)propionitrile (DPN) was purchased from Cayman Chemical, Ann Arbor, MI, USA/Funakoshi 10008842. The Hep G2 cell line from human hepatocellular carcinoma cell line was purchased from the American Type Culture Collection (HB-8065; ATCC, Manassas, VA, USA).
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