Ovcar3

Manufactured by Thermo Fisher Scientific

Sourced in United States, China, Canada, France, United Kingdom

The OVCAR3 is a cell line derived from a human ovarian adenocarcinoma. It is commonly used in cancer research, particularly for the study of ovarian cancer.

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109 protocols using ovcar3

Overexpression and Knockdown of Sam68 in OVCAR-3 Cells

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The human EOC cell line OVCAR-3 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). OVCAR-3 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Carlsbad, CA, USA), which was supplemented with 10% fetal bovine serum (FBS) (Gibco, Rockville, MD, USA) and 1% streptomycin/penicillin (Sigma-Aldrich, St. Louis, MO, USA) in an incubator with 5% CO₂ at 37°C. The medium was changed on alternate days and cells were split before they reached more than 90% confluence. Expression vectors pRC/CMV-Sam68-FLAG (Sam68 Up) and pcRC/CMV (Con Up, as control) were purchased from Promega (Promega, Madison, WI, USA). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was utilized to transfect plasmids into OVCAR-3 cells. Sam68 knockdown was conducted by using siRNAi-Sam68 (5′-tga gtt aaa ata gat tta gga a-3′) (as control siRNA, 5′-uuc uca gaa cgu gug acg u-3′), which were transfected into OVCAR-3 cells with Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA, USA).

Dong L., Che H., Li M, & Li X. (2016). Sam68 is Overexpressed in Epithelial Ovarian Cancer and Promotes Tumor Cell Proliferation. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 22, 3248-3750.

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Cisplatin-Resistant Ovarian Cancer Cell Lines

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The human high-grade serous ovarian cancer cell lines OV-90 and OVCAR3 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). OV-90CIS and OVCAR3CIS cells were generated by exposing parental cell lines to increasing doses of cisplatin as previously described [70 (link)]. OV-90 and OV-90CIS cells were maintained in M199 (Gibco, Thermo Fisher Scientific, Grand Island, NY, USA)/MCDB-105 (Sigma-Aldrich, St. Louis, MO, USA) media. OVCAR3 and OVCAR3CIS cells were maintained in RPMI-1640 (Thermo Scientific, Grand Island, NY, USA) medium supplemented with 0.01 mg/mL insulin (Sigma-Aldrich). In all cases, the medium was supplemented with 10% fetal bovine serum (FBS; HyClone, GE Healthcare Life Sciences, Logan, UT, USA) and 0.1% antibiotic/antimycotic solution (HyClone). The cells were incubated at 37 °C in 5% CO₂ with 95% air. All cell lines were screened for mycoplasma using the LookOut^® Mycoplasma PCR detection kit (Sigma). In vitro experiments were performed at 70–85% cell confluence.

Flores-Colón M., Rivera-Serrano M., Reyes-Burgos V.G., Rolón J.G., Pérez-Santiago J., Marcos-Martínez M.J., Valiyeva F, & Vivas-Mejía P.E. (2024). MicroRNA Expression Profiles in Human Samples and Cell Lines Revealed Nine miRNAs Associated with Cisplatin Resistance in High-Grade Serous Ovarian Cancer. International Journal of Molecular Sciences, 25(7), 3793.

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Investigating BMPER Expression in Ovarian Cancer

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Human ovarian cancer cell lines (CAOV3, OVCAR3, SKOV3, and ES-2) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). CAOV3, OVCAR3, and SKOV3 cells were incubated in 37°C and 5% CO2 incubator with RPMI 1640 medium containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), and ES-2 cells were cultured in the same incubator in McCoy's 5A medium containing 10% fetal bovine serum. BMPER was expressed in four ovarian cancer cell lines: CAOV3, OVCAR3, SKOV3, and ES-2. Among these cell lines, the miRNA and protein expression levels of BMPER were higher in CAOV3 and OVCAR3 cells, so these two cell lines were selected for the RNA interference experiment. CAOV3 and OVCAR3 cells were transfected with BMPER-specific siRNA and negative control (NC) using Lipofectamine 3000 (Thermo Fisher Scientific, USA). Cells were collected 72 hours after transfection, and the effects on BMPER interference were detected by RT-PCR and Western blot. The BMPER siRNA sequence (GenePharma, Shanghai, China) is as follows: sense, GGUCCUGUGUGACAGACAUTT and antisense, AUGUCUGUCACACAGGACCTT.

Xi Y., Nie X., Wang J., Gao L, & Lin B. (2020). Overexpression of BMPER in Ovarian Cancer and the Mechanism by which It Promotes Malignant Biological Behavior in Tumor Cells. BioMed Research International, 2020, 3607436.

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Profiling Human Cancer Cell Lines

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The human colorectal HCT 116 (CVCL_0291), Caco-2(CVCL_0025), HT-29 (CVCL_0320), SW480 (CVCL_0546), SW620 (CVCL_0547), breast MCF-7(CVCL_0031), T-47D (CVCL_0553), MDA-MB-231(CVCL_0062), ovarian Caov-3(CVCL_0231), SK-OV-3(CVCL_0532) cancer cell lines and HEK293T(CVCL_0063) were obtained from the GeneChem Corporation (Shanghai, China). OVCAR-3(CVCL_0465) was obtained from the Nanjing KeyGen Biology (Nanjing, China). All human cell lines have been authenticated using STR profiling. OVCAR-3, Caco-2, MCF-7, Caov-3 and HEK293T cells were cultured in DMEM (Gibco, Carlsbad, CA, USA). T-47D and OVCAR-3 cells were cultured with RPMI 1640 (Gibco, Carlsbad, CA, USA). HCT 116 and HT-29 cells were cultured with McCoy’s 5a (Gibco, Carlsbad, CA, USA). SW480, SW620, and MDA-MB-231 cells were cultured with Leibovitz’s L-15 (Gibco, Carlsbad, CA, USA) medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. To inhibit NF-κB activities, BAY 117082 (Selleckchem, Houston, TX, USA) was used.

Zhao L., Jiang L., Zhang M., Zhang Q., Guan Q., Li Y., He M., Zhang J, & Wei M. (2021). NF-κB-activated SPRY4-IT1 promotes cancer cell metastasis by downregulating TCEB1 mRNA via Staufen1-mediated mRNA decay. Oncogene, 40(30), 4919-4929.

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Ovarian Cancer Cell Line Maintenance

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Ovarian cancer cell lines, OVCAR3, OV-90, PEO1 and PEO4 were purchased from ATCC (American Type Culture Collection) and were maintained, respectively, in RPMI (OVCAR3, PEO1 and PEO4) and DMEM (OV-90) (Gibco, Paisley, UK), supplemented with 20% (OVCAR-3) or 10% (OV-90, PEO1 and PEO4) fetal bovine serum (FBS; Gibco BRL, Italia), 1% L- Glutamine and 1% of penicillin – streptomycin (Gibco, Paisley, UK). Kuramochi cells were purchased from Sekisui XenoTech, LLC and were maintained in RPMI (Gibco, Paisley, UK), supplemented with 10% FBS, 1% L-Glutamine and 1% of penicillin – streptomycin (Gibco, Paisley, UK). Olaparib (AZD2281), and P005091 were provided by SelleckChem. PARG inhibitor (PDD00017273), SB-216763, cycloheximide, MG132 and cisplatin were provided by Merk Millipore. The genetic background of the ovarian cancer cells is reported in Additional File 1: Table S1.

Morra F., Merolla F., Damia G., Ricci F., Varricchio S., Ilardi G., Arenare L., Califano D., Napolitano V., Fruscio R., Melillo R.M., Palazzo L, & Celetti A. (2022). The disruption of the CCDC6 – PP4 axis induces a BRCAness like phenotype and sensitivity to PARP inhibitors in high-grade serous ovarian carcinoma. Journal of Experimental & Clinical Cancer Research : CR, 41, 245.

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Cell Line Cultivation and Maintenance

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SKOV3, OVCAR3, MCF7, MDA231, T47D were obtained from the ATCC (Manassa, Virginia, USA). SKOV3 (ATCC-HBT-77) was grown in McCOY's 5a (Life technologies, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (FBS, Wisent technologies, St-Bruno, Quebec
Canada). MCF7 (ATCC-HTB-22) and MDA231 (ATCC-HTB-26) cells were grown in DMEM/F12 (Life technologies), supplemented with 10% FBS. T47D (ATCC-HTB-133) and OVCAR3 (HTB-161) cells were grown in RPMI-1640 (Life technologies), supplemented with 10% and 20% FBS, respectively. Culture method was followed as described by ATCC for each cell line. Kuramochi (JCRB No. JCRB0098) and OVSAHO (JCRB No. JCRB1046) cells were obtained from JCRB (Japanese Collection of Research Bioresources) Cell Bank and Sekisui Xenotech LLC (Cambridge, Kansas City, USA). Cells were grown in RPMI-1640 (Life technologies) supplemented with 10% FBS.

Sowamber R., Chehade R., Bitar M., Dodds L.V., Milea A., Slomovitz B., Shaw P.A, & George S.H. (2019). CCAAT/enhancer binding protein delta (C/EBPδ) demonstrates a dichotomous role in tumour initiation and promotion of epithelial carcinoma. EBioMedicine, 44, 261-274.

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Ovarian Cancer Cell Line Maintenance

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The human ovarian cancer cell line A2780 was purchased from European Collection of Authenticated Cell cultures (ECACC, Salisbury, UK). OVCAR3 and OV90 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). ES-2 cells were generously provided by Dr H. Albrecht (University of South Australia). All cell lines were verified by short tandem repeat (STR) testing in 2021 (Promega GenePrint^®10; Griffith University DNA sequencing facility, QLD, Australia). A2780, ES-2 and OVCAR3 cell lines were maintained in RPMI-1640 media (11,875,093, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Bovogen Biologicals, Melbourne, Vic, Australia) and antibiotics (100U penicillin G, 100 µg/mL streptomycin sulphate and 0.25 µg/mL amphotericin B, Sigma-Aldrich, St Louis, MO, USA) and maintained at 37 °C in 5% CO₂ environment.

Price Z.K., Lokman N.A., Sugiyama M., Koya Y., Yoshihara M., Oehler M.K., Kajiyama H, & Ricciardelli C. (2023). Disabled-2: a protein up-regulated by high molecular weight hyaluronan has both tumor promoting and tumor suppressor roles in ovarian cancer. Cellular and Molecular Life Sciences, 80(11), 320.

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Culturing Human Ovarian Cancer Cell Lines

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The human EOC cell lines OVCAR-3 (HTB-161) and SK-OV-3 (HTB-77) were purchased from ATCC (Wesel, Germany) and cultured at 37°C in a 5% CO₂-containing humidified atmosphere. The SK-OV-3-Luc IP1 cell line, a more aggressive, luciferase positive OC cell line compared to the SK-OV-3 cell line, was created through in vivo selection and cultured at 37°C in a 10% CO₂-containing humidified atmosphere. Short tandem repeat profiling was conducted as previously described.21 (link) The SK-OV-3 cell line and SK-OV-3 Luc IP1 cell lines were cultured both in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies, Merelbeke, Belgium), while the OVCAR-3 cell line was cultured in RPMI 1640 Medium supplemented with Gibco GlutaMAX^TM (Life Technologies, Merelbeke, Belgium). All mediums were supplemented with 2% penicillin/streptomycin (Life Technologies) and 10% FCS (Sigma-Aldrich, Overijse, Belgium).

Demuytere J., Carlier C., Van de Sande L., Hoorens A., De Clercq K., Giordano S., Morosi L., Matteo C., Zucchetti M., Davoli E., Van Dorpe J., Vervaet C, & Ceelen W. (2024). Preclinical Activity of Two Paclitaxel Nanoparticle Formulations After Intraperitoneal Administration in Ovarian Cancer Murine Xenografts. International Journal of Nanomedicine, 19, 429-440.

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Ovarian Cancer Cell Line Characterization

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Human EOC cell line JHOC-5 was kindly provided by Dr Kazunori Ochiai (Jikei University, Tokyo, Japan). Cell lines RMG-I, RMG-II, RMG-V, RMUG-L, and RMUG-S were established and maintained at our laboratory. JHOS-2 was purchased from RIKEN Bioresource Center Cell Bank (Tsukuba, Japan). OVCAR3 and OVK18 were purchased from the American Type Culture Collection (Rockville, MD, USA). Cell lines JHOC-5, RMG-I, RMG-II, and RMG-V were clear-cell subtypes, JHOS-2 and OVCAR3 were serous subtypes, RMUG-S and RMUG-L were mucinous subtypes and OVK18 was endometrioid subtype. Cells were grown in Dulbecco's modified Eagle's medium (DMEM)/F12 (JHOC-5, RMG-II, JHOS-2, RMUG-L, and RMUG-S), RPMI-1640 (OVCAR3 and OVK18), or F12 (RMG-I) supplemented with 10% heat-inactivated FBS, 100 μg ml⁻¹ streptomycin (Life Technologies Inc., Grand Island, NY, USA), and 100 IU ml⁻¹ penicillin (Life Technologies Inc.). The cell lines used in our study were authenticated by short tandem repeat profiling on January 2012.

Nishio H., Yaguchi T., Sugiyama J., Sumimoto H., Umezawa K., Iwata T., Susumu N., Fujii T., Kawamura N., Kobayashi A., Park J., Aoki D, & Kawakami Y. (2014). Immunosuppression through constitutively activated NF-κB signalling in human ovarian cancer and its reversal by an NF-κB inhibitor. British Journal of Cancer, 110(12), 2965-2974.

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OVCAR-3 Glycoproteomic Analysis

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Human ovarian carcinoma cell line OVCAR-3 (ATCC, HTB161) was obtained from ATCC (Rockville, MD). The cell line was tested for mycoplasma contamination by ATCC before being used in experiments. A total of 32 dishes (15 cm × 15 cm) of OVCAR-3 cells were cultured in RPMI 1640 medium with 10% FBS to 50% confluence, and half of the dishes were treated with 1 µM tunicamycin for 48 h to inhibit N-glycosylation. In order to analyze the glycoprotein changes in tunicamycin-treated cells, the OVCAR-3 cells were also treated with 1 µM tunicamycin in triplicate and collected at six different time points (0, 6, 12, 24, 48 and 72 h). Separate dishes of OVCAR-3 cells were cultured in SILAC RPMI 1640 medium containing heavy isotope-labeled ¹³C₆-l-lysine and ¹³C₆¹⁵N₄-l-arginine (Cambridge Isotope Laboratories, Andover, MA) supplemented with 10% dialyzed FBS (diFBS) (Invitrogen, Carlsbad, CA) to generate heavy SILAC-labeled (K6, R10) OVCAR-3 cells. The cells were cultured for approximately ten doublings in the SILAC medium to ensure complete labeling³¹. After removing the medium, the cells were washed five times with phosphate buffered saline (PBS, pH 7.4) buffer and then lysed directly with 8 M urea/1 M NH₄HCO₃ solution³². Lysates were briefly sonicated until the solutions were clear. Protein concentrations were determined by BCA protein assay reagent (Thermo Scientific, Fair Lawn, NJ).

Sun S., Shah P., Eshghi S.T., Yang W., Trikannad N., Yang S., Chen L., Aiyetan P., Höti N., Zhang Z., Chan D.W, & Zhang H. (2015). Comprehensive analysis of protein glycosylation by solid-phase extraction of N-linked glycans and glycosite-containing peptides. Nature biotechnology, 34(1), 84-88.

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