Human NSCLC cell lines (H226, H460, and H1299) and the normal lung cell line (BEAS-2B) were purchased from the ATCC. The cell lines were cultured as described in our previous studies.14 (link),41 (link),42 (link),43 (link) WEB2086, a PAFR antagonist, was purchased from Sigma-Aldrich (Sigma-Aldrich).
Web2086
Manufactured by Merck Group
Sourced in Sao Tome and Principe
The WEB2086 is a laboratory equipment product manufactured by Merck Group. It is designed to perform specific laboratory functions. No further details are available without the risk of unintended interpretation or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin sulfate, by Merck Group (3 mentions)
Penicillin g, by Merck Group (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Ag1478, by Merck Group (2 mentions)
Beas 2b, by American Type Culture Collection (1 mentions)
H1299, by American Type Culture Collection (1 mentions)
Alexa fluor 488 annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (1 mentions)
A cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Sodium azide nan3, by Fujifilm (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Opti mem 1 medium, by Thermo Fisher Scientific (1 mentions)
Pd98059, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Foetal calf serum, by Thermo Fisher Scientific (1 mentions)
5 protocols using web2086
1
NSCLC Cell Lines and PAFR Antagonist
2
Ovarian Cancer Cell Lines Experiments
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The ovarian cancer cell lines CAOV-3 and SKOV-3 (purchased from the Cell Bank of the Chinese Academy of Science, Shanghai, China) were cultured at 37°C in a humidified 5% CO2 atmosphere in RPMI-1640 medium with 10% fetal calf serum (Gibco, Invitrogen, Carlsbad, CA), 100 IU/ml penicillin G, and 100 mg/ml streptomycin sulfate (Sigma-Aldrich, St. Louis, MO). AG1478 (EGFR inhibitor)
[24 (link)] and WEB2086 (PAFR antagonist)
[25 (link)] were purchased from Sigma-Aldrich (St Louis, MO). A Cell Counting Kit 8 (CCK8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan), and the Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit was obtained Invitrogen (Carlsbad, CA). Rabbit polyclonal antibodies directed against PAFR, cleaved-caspase3, cleaved-PARP, phospho/total- EGFR, phospho/total- β-arrestin2, phospho/total- P70S6K, phospho/total- AKT, phospho/total- 4EBP1, and phospho/total- ERK were used in this study. All of these antibodies were purchased from Cell Signaling Technology Co. Mouse monoclonal antibodies directed against actin were also used (Sigma, Missouri, USA).
[24 (link)] and WEB2086 (PAFR antagonist)
[25 (link)] were purchased from Sigma-Aldrich (St Louis, MO). A Cell Counting Kit 8 (CCK8) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan), and the Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit was obtained Invitrogen (Carlsbad, CA). Rabbit polyclonal antibodies directed against PAFR, cleaved-caspase3, cleaved-PARP, phospho/total- EGFR, phospho/total- β-arrestin2, phospho/total- P70S6K, phospho/total- AKT, phospho/total- 4EBP1, and phospho/total- ERK were used in this study. All of these antibodies were purchased from Cell Signaling Technology Co. Mouse monoclonal antibodies directed against actin were also used (Sigma, Missouri, USA).
3
Preparation of Oxidative Stress Modulators
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EP powder was purchased from Waken Btech, Co., Ltd. (Kyoto, Japan), and the solution was prepared by diluting the powder in 0.1% dimethyl sulfoxide (DMSO; FUJIFILM Wako Pure Chemical Industries, Osaka, Japan). To prepare inactive EP (negative control), 1 mM EP solution was incubated at 37°C with a humidified atmosphere of 5% CO2 (Thermo Fisher Scientific, San Jose, CA) for 24 h. Catalase (CAT; FUJIFILM Wako Pure Chemical Industries), and superoxide dismutase (SOD; Nippon Kayaku Co., Tokyo, Japan) were dissolved in distilled water to a final concentration of 10,000 U/ml, aliquoted, and stored at 4°C. WEB2086 (Sigma-Aldrich, St. Louis, MO) was prepared by dissolving 1 mg in 220 μl of DMSO then 1.98 ml of PBS (−) was added to reach a final concentration of 10 mM (and a DMSO concentration of 10%). Sodium azide (NaN3) was obtained from FUJIFILM Wako Pure Chemical Industries and dissolved in medium to prepare the desired final concentration. 0.143 mg of MnTMPyP (Calbiochem, Merck KGaA, Darmstadt, Germany) was dissolved in medium to a final concentration of 10 μM for all experiments, and the remaining solution was aliquoted and stored at −20°C. Effective concentrations and incubation times for inhibitors, ROS scavengers, et cetera, were decided in consideration of toxicity using cell viability assay.
4
Ovarian Cancer Cell Line Maintenance and Transfection
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The ovarian cancer cell lines CAOV3 and SKOV3 (obtained from the Cell Bank of the Chinese Academy of Science, Shanghai, China) were maintained at 37°C in a humidified 5% CO2 atmosphere in RPMI-1640 medium with 10% fetal calf serum (Gibco, Invitrogen, Carlsbad, CA), 100 IU/ml penicillin G, and 100 mg/ml streptomycin sulfate (Sigma-Aldrich, St. Louis, MO). Cells were serum starved by incubation in serum-free medium for 12–24 hours before the start of the experiments. Lipofectamine 2000 Transfection Reagent and Opti-MEM-1 Medium (Invitrogen, Carlsbad, CA) were used for plasmid and siRNA transfection. The vector encoding cPLA2 and cPLA2-targeted siRNA were synthesized by Shanghai GenePharma Co. AG1478 (EGFR inhibitor) [20 (link)] and WEB2086 (PAFR inhibitor) were purchased from Sigma-Aldrich (St Louis, MO). PD98059 (ERK inhibitor) and LY294002 (PI3K inhibitor) were obtained from Cell Signaling Technology (Boston, MA). Rabbit polyclonal antibodies that were used in this study were directed against phospho/total-EGFR, phospho/total-PLCβ, phospho/total-cPLA2, phospho/total-Akt, and phospho/total-ERK. All the antibodies were purchased from Cell Signaling Technology Co. The mouse monoclonal antibodies that were used in this study were directed against actin (Sigma, Missouri, USA).
5
Culturing Ovarian Cancer Cell Lines and Mesenchymal Stem Cells
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The OCC lines OVCA433, RMUG-L, 3AO, OMC685, TOV112D, and DOV13 were kindly provided by Bin Ye (Department of Obstetrics and Gynecology and Reproductive Biology, Laboratory of Gynecologic Oncology and Epidemiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA). These cell lines, along with SKOV3 and ES-2 cells (purchased from the Cell Bank of the Chinese Academy of Science, Shanghai, China), were cultured at 37 °C in a humidified 5% CO2 atmosphere in Roswell Park Memorial Institute-1640 (RPMI-1640) medium with 10% foetal calf serum (Gibco, Invitrogen, Carlsbad, CA) (serum-free medium was used when measuring the concentration of PAF in the medium), 100 IU/ml penicillin G, and 100 mg/ml streptomycin sulfate (Sigma-Aldrich, St. Louis, MO). MSCs (purchased from Cyagen Biosciences, Guangzhou, China) were cultured at 37 °C in a humidified 5% CO2 atmosphere in adult bone marrow mesenchymal stem cell (MSC) complete medium. PAF, ginkgolide B (GB), and WEB2086 (PAFR antagonist) were purchased from Sigma-Aldrich. Human and mouse PAF enzyme-linked immunosorbent assay (ELISA) kits were purchased from Groundwork Biotechnology Diagnosticate (San Diego, CA). The stem cell induction differentiation kit was purchased from Cyagen Biosciences (Guangzhou, China).
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