The human PCa cell lines PC3 and DU145 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in RPMI-1640 medium (Gibco-BRL; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco-BRL; Thermo Fisher Scientific) at 37°C under 5% CO2 in a humidified incubator. For cell treatment, PC3 and DU145 cells were cultured in RPMI-1640 medium supplemented with genistein (purity >98%, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and AG1024 (Sigma-Aldrich; Merck KGaA) at series concentrations. genistein (0, 10, 20, 30, 50 and 100 µM) and AG1024 (10 µM) (5 (link)) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA). X-ray irradiation was delivered using an X-6 MV photon linear accelerator (Varian Associates Inc., Palo Alto, CA, USA) at room temperature with a dose rate of 2 Gy/min (25 (link)). Cells treated with DMSO were considered as negative control for irradiation or drug treatment.
Ag1024
Manufactured by Merck Group
Sourced in United States, Germany
AG1024 is a laboratory equipment product manufactured by Merck Group. It is designed to perform specific laboratory functions. The core function of AG1024 is to serve as a tool for researchers and scientists in their work. No further details about the intended use or interpretation of the product's capabilities are provided.
Automatically generated - may contain errors
Lab products found in correlation
Anti β catenin, by Santa Cruz Biotechnology (2 mentions)
Pa5 37602, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Pertussis toxin, by Merck Group (1 mentions)
Antilumican, by Santa Cruz Biotechnology (1 mentions)
Anti erk1 2, by Santa Cruz Biotechnology (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti igf ir, by Santa Cruz Biotechnology (1 mentions)
U0126, by Cell Signaling Technology (1 mentions)
Keratanase 2, by Seikagaku (1 mentions)
Genistein, by Merck Group (1 mentions)
Gibco brl, by Thermo Fisher Scientific (1 mentions)
Du145, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Du145, by American Type Culture Collection (1 mentions)
Daunorubicin, by Merck Group (1 mentions)
11 protocols using ag1024
1
Genistein and AG1024 Effects on Prostate Cancer Cells
2
Cytotoxicity Screening of Pharmacological Agents
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U0126, AG1024, LY294002, vincristine, and daunorubicin were dissolved in dimethylsulfoxide (DMSO) and dexamethasone in 100% ethanol at stock concentrations of 10mmol/L. Cells were incubated with U0126, AG1024, vincristine, daunorubicin, or dexamethasone (Sigma-Aldrich, St. Louis, MO, USA) at the indicated doses at 37°C for 72hours and subsequently reacted with Cell TitreGlo reagent according to the manufacturer's instructions (Promega). Luminescence was quantified using a Victor Wallac plate reader.
3
Purification and Characterization of HIV Proteins
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All media, sera, and supplements were obtained from Thermo Fisher Scientific (Grand Island, NY) unless otherwise noted. All primary antibodies and LY294002 were obtained from Cell Signaling Technology ([CST], Beverly, MA). Fluconazole was obtained from Spectrum Chemicals (New Brunswick, NJ). AG1024, 3-nitroproprionic acid, pertussis toxin, and all other chemicals were obtained from Sigma (Saint Louis, MO), unless otherwise noted. Purified endotoxin-free recombinant HIV Tat (1–72) was made and characterized as previously described [9 (link), 10 (link)]. Gp120 derived from HIVIIIB and HIVSF was acquired from the NIH-AIDS repository (Bethesda, MD, USA). HIV-gp120 transgenic mice were kindly provided by Dr. Mucke at the Gladstone Institute.
4
Investigating IGF-I and TGF-β2 Signaling Pathways
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Recombinant human insulin-like growth factor I (IGF-I; 291-G1; 10 ng/ml) and transforming growth factor-β2 (TGF-β2; 302-B2-010; 10 ng/ml) were obtained from R&D Systems. A selective inhibitor of ERK1/2 (U0126; 10 μM; Cell Signaling Technology, Inc.) and allosteric inhibitor of IGF-IR (AG1024; 1 µM; Sigma-Aldrich; Merck KGaA) were used in the present study for 1 h. Primary antibodies from Santa Cruz Biotechnology, Inc. were used, and these included anti-lumican (sc166871; mouse monoclonal; 1/100 dilution for western blot analysis or 1/50 for immunofluorescence), anti-β-catenin (sc7963; mouse monoclonal; 1/300 dilution), anti-ERK1/2 (sc514302; mouse monoclonal; 1/200 dilution), anti-IGF-IR (sc81464; mouse monoclonal; 1/100 dilution), anti-pERK1/2 (sc136521; mouse monoclonal; 1/100 dilution), anti-Smad2 (sc6200; goat polyclonal; 1/200 dilution) and anti-pSmad2 (sc101801; rabbit polyclonal; 1/200 dilution). In addition, anti-actin (MAB1501; mouse monoclonal; 1/5,000 dilution; EMD Millipore), anti-p-IGF-IR (PA5-37602; polyclonal rabbit; 1/500 dilution; Thermo Fisher Scientific, Inc.), keratan sulfate (KS; 270427; mouse monoclonal; 1/1,000 dilution; Seikagaku Corporation) and keratanase II (100812; 0,005 µ/ml; Seikagaku Corporation) were utilized. Secondary-HRP antibodies anti-rabbit (AP182PR) and anti-mouse (AP192PM) were used at a 1/5,000 dilution and obtained from Millipore.
5
Sprague-Dawley Rat Astrocyte and Neuron Cultures
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We used postnatal Sprague-Dawley rats for in vitro cultures (P3 days for astrocytes and E18 for neurons). 40 C57*B6 mice at 6–8 weeks old were used for in vivo study (10 for each group). All experiments were carried out based on medical ethics guidelines (20170223-001). KA and AG1024 (IGF-1R antagonist) were purchased from Sigma (Steinheim, Germany).
6
IGF-I Stimulated DNA Synthesis Assay
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DNA synthesis was assessed by measuring the incorporation of 5-bromo-2-deoxyuridine (BrdU) into DNA [13 (link)]. Briefly, cells were seeded in 96-well culture plates (1200 cells/well), and maintained in medium containing varying doses of IGF-I for 24h at 37° C. BrdU or phosphate buffer saline (as background control) was added to each well during the final 8h of treatment. After fixation, cells were incubated with anti-BrdU antibody for 1h at room temperature, and with substrate solution for 15 min in the dark at room temperature. The signals were quantitated using a spectrophotometric plate reader set at wavelength of 450/540 nm. To examine if high dose IGF-I also activated the other signaling pathways to stimulate DNA synthesis, cells were treated with IGF-I and AG1024 (Sigma Aldrich, MO, USA) concomitantly.
7
Biglycan and IGF-I Signaling Pathway
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Recombinant human biglycan (2667-CM) and IGF-I (insulin-like growth factor I; 291-G1) were obtained from R&D Diagnostics. Selective inhibitor of ERK1/2 (Cell Signaling Technology; U016) and allosteric inhibitor of IGF-IR (Sigma-Aldrich; AG1024) were used in this study. Primary antibodies from Santa Cruz Biotechnology used, anti-biglycan (sc100857; mouse monoclonal; 1/100 dilution), anti-β catenin (sc7963; mouse monoclonal; 1/300 dilution and 1/50 for immunofluorescence experiments), anti-IGF-IR total (sc81464; mouse monoclonal; 1/100 dilution), anti-pERK1/2 (sc136521; mouse monoclonal; 1/100 dilution) and anti-fibrillarin (sc374022; 1/100 dilution). In addition, anti-actin (Millipore MAB1501; mouse monoclonal; ,500 dilution), anti-ERK total (Thermo scientific MA5-15343; mouse monoclonal; 1/500 dilution), anti-pIGF-IR (Thermo scientific PA5-37602; polyclonal rabbit; 1/500 dilution for western blot and 1/50 for immunofluorescence experiments), anti-tubulin (Sigma-Aldrich T4026; monoclonal mouse; 1/1,000 dilution) and anti-LRP6 (Elabscience E-AB17347; rabbit polyclonal; 1/50 dilution) were utilized. Secondary-HRP antibodies anti-rabbit (AP182PR); and anti-mouse (AP192PM) were used in a 1/3,000 dilution and obtained from Millipore.
8
Humanized Monoclonal Antibodies and IGF1R Inhibitors
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MK-0646 (Dalotuzumab; Merck, Sharp and Dohme Ltd., Whitehouse Station, NJ, USA) and A12 (Cixutumumab; ImClone Systems, New York, NY, USA) are humanized IgG1 monoclonal antibodies antagonist to the human IGF1R [60 (link), 61 (link)]. The antibodies were diluted in 20 mM histidine and 150 μM NaCl and used at a concentration of 10 mg/ml. AEW541 (Novartis Pharma, Basel, Switzerland) and AG1024 (Sigma-Aldrich Ltd, St. Louis, MO, USA) are selective IGF1R tyrosine kinase inhibitors. AEW541 and AG1024 were kept as a stock solution (10 mM) in DMSO and stored at –20° C. AEW541 is a reversible, ATP-competitive phosphorylation inhibitor that exhibits high selectivity towards IGF1R over insulin receptor [62 (link)]. AG1024 belongs to the tyrphostin family of tyrosine kinase inhibitors [63 (link)].
9
Recombinant IGF-I and IGF-IR Inhibitor
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Human, recombinant IGF-I was purchased from Gropep (Adelaide, Australia) and an IGF-IR inhibitor, AG1024, was obtained from Sigma-Aldrich, Dorset, UK.
10
Reagents for Cell Culture Experiments
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Cell culture media (DMEM/F12, RPMI-1640, phenol red-free RPMI-1640), FBS, and charcoal-stripped FBS were purchased from Thermo Fisher Scientific. The IGF-1 receptor antagonist AG 1024 was purchased from EMD Millipore. The SCD-1 inhibitor A939572 was purchased from Biovision. 17β-estradiol (17β-ED), IGF-1, 4-OH tamoxifen, and DMSO were purchased from Sigma-Aldrich. 17β-ED and 4-OH tamoxifen were dissolved in ethanol, IGF-1 was prepared in sterile water and both A939572 and AG 1024 were prepared in DMSO.
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