Characterization of Ovarian Cancer Cell Lines

Kuramochi, SNU119, OV90, TOV112D, OVCA429, OVCAR8, and SKOV3-ip cells have been previously described and cell passaging was never exceeded eighteen (18 (link), 19 (link)). The cell lines obtained from NCI, ATCC, and Seoul National University were authenticated at IDEXX Bioresearch (Columbia, MO) using nine human short tandem repeat profile (20 (link)). Cells were monitored for mycoplasma contamination using previously published PCR-based protocol (21 (link)). Patient derived cell line ASC022315, ASC022415, ASC031915 were isolated from the ascites samples of patients at the Stephenson Cancer Center, University of Oklahoma Health Science Center, Oklahoma City, OK, USA. The study was approved by the OUHSC Office of Human Research Participant Protection (HRPP) Institutional Review Board (IRB) and samples were collected with the informed consent from the patients. The ascites derived ovarian cancer cells were maintained in MCDB:DMEM (1:1) supplemented with 10% FBS and 50 µg/mL streptomycin. For serum-starvation, the above media without serum was supplemented with 0.1% BSA Fraction V, heat-shock, fatty acid ultra-free (Roche, Indianapolis, IN), 50 U/mL penicillin and 50 µg/mL streptomycin (Mediatech). Lysophosphatidic acid (1-oleoyl-2-hydroxy-sn-glycero-3-phosphate) was obtained from Avanti Polar Lipids (Alabaster, AL) and dissolved into 10 mM stock solutions in PBS with 0.1% BSA and stored at −80°C until use. siRNAs against HIF1α, Gαi2, Rac1, Rac2, Rac3, Nox1, Nox2, Nox3, Nox4, Nox5, HKII, and the control ON-TARGETplus Non-targeting siRNA #1 were purchased from Dharmacon (Lafayette, CO). shRNA targeting Gα₁₂, Gα₁₃, Gα_q, Gα_i2 and nonsense shRNA as well as immunoblot analyses and quantifications of immunoreactive bands have been previously described and used (22 (link)). N-acetyl-L-cysteine (NAC) and Bromopyruvic acid (3-Bromo-2-oxpropionic acid) was obtained from Sigma (St. Louis, MO). Apocynin, and Ki16425 inhibitor was procured from Tocris Bioscience (Bristol, UK). The Rac1 inhibitor NSC23766 was from EMD Millipore (Billerica, MA). The HIF1α inhibitor, PX-478 was obtained from MedKoo Biosciences (Morrisville, NC).