Tov 21g

The ovarian tumour cell lines A2780 (obtained from Dr. Ozols, Bethesda, MD), OVCAR-5 (obtained by Dr. Camalier, NCI-NIH) and TOV-21G (purchased from ATCC, Manassas, VA), were cultured in RPMI 1640 (Lonza, Basel, Switzerland) containing 10% foetal calf serum at 37°C under 5% CO₂. To establish stable transfectants, A2780 cells, OVCAR-5, and TOV-21G were seeded (5 × 10⁵), in 60 mm dishes and 24 h later, a mixture of Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) and 8 μg of expression or silencing plasmid was added and incubated for 6 h. Cells were subsequently cultured in medium supplemented with 10% serum for an additional 48 h before adding G418 (Gibco Brl, Paisley, UK) at a concentration of 400 μg/ml for A2780 and OVCAR-5 cells or Puromycin (Sigma Aldrich, St Louis, MO) at a concentration of 2.5 μg/ml for TOV-21G, used for the selection of transfectant clones. miR-200c and miR-141 mimics were transiently transfected in A2780 and TOV-21G cells. Cells were seeded and transfected according to the above mentioned transfection protocol using 20 μM miR-200c mimic (Thermo Fisher) and 20 μM miR-141 mimic (Thermo Fisher) and analyzed after 72 hours.

All of the human ovarian cancer cell lines used in our study, including TOV-21G, IGROV-1, SKOV3, and A2780, were obtained from the American Type Culture Collection (ATCC, Manassas, VA). TOV-21G cells were maintained in MCDB105 and M199 (1:1) supplemented with 15% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA). The IGROV-1 cell line was maintained in RPMI1640 supplemented with 10% FBS. The SKOV3 cell line was maintained in DMEM/F-12 with 10% FBS. The A2780 cell line was maintained in RPMI1640 with 10% FBS, 0.1 mM MEM non-essential amino acids, and 10 mM sodium pyruvate. All of the culture media contained 5 mM L-glutamine and antibiotics. All of the human ovarian cancer cell lines were incubated at 37°C, in a humid 5% CO₂ atmosphere.

LECs were purchased from ScienCell (San Diego, California, USA)and were grown in Endothelial Cell Medium (ECM) according to the manufacturer's instruction. Human EOC cell lines SKOV3, TOV-112D, TOV-21G, A2780, OV-90, OVCAR3 and Caov3 were purchased from American Type Culture Collection (Rockville, MD, USA). SKOV3 cells were cultured in McCoy's 5A medium supplemented with 10% foetal bovine serum (FBS). OVCAR3 cells were cultured in DMEM supplemented with 10% FBS and 0.01 mg/ml bovine insulin. Caov3 cells were cultured with DMEM containing 10% FBS. TOV-112D, TOV-21G and OV-90 cells were cultured in a 1:1 mixture of medium 199 (Invitrogen, Carlsbad, CA) and medium 105 (Sigma-Aldrich, St Louis, MO) with 10% FBS. NOFs were obtained from normal ovarian tissues, and CAFs were obtained from ovarian tumour tissues following procedures as previously described [31 (link)]. All these cell lines were grown in a humidified atmosphere with 5% CO₂ at 37°C.

The 293 normal human embryonic kidney cells and TOV-21G and OV-90 ovarian cancer cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in Eagle’s minimum essential medium (293 cells) and Roswell Park Memorial Institute (RPMI)-1640 medium (TOV-21G and OV-90) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen Corp, Carlsbad, CA, USA). All culture media and FBS were from Welgene (Daegu, Korea). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂. Media were routinely changed after every 3 d.