Fetal bovine serum (fbs)

Epithelial ovarian cancer cell lines A2780 (from chemonaive primary tumor) and SKOV-3 (from ascites fluid) were maintained in RPMI (HyClone, Thermo Scientific, Stockholm, Sweden), supplemented with 10% fetal bovine serum (HyClone) and 2.5 μg/mL Plasmocin (InvivoGen, Toulouse, France) at 37 °C under 5% CO₂ in humidified incubators. The identities of both cell lines were validated during 2012 using short-tandem repeat analysis AmpFlSTR Identifiler kit (Applied Biosystems/Life Technologies, Stockholm, Sweden).^{39 (link)}

The siRNAs (80 nM) for CDK4, GABPB1, TRPM7, USP8, SPPL2A, and control siRNA (IDT) were transfected into the Bear cell line using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA), according to the manufacturer’s recommendations (Tables S1 and S3). CDK4 siRNA was used as the positive control. The siRNAs (10 nM) for GABPB1, TRPM7, and SPPL2A, as well as the control siRNA (IDT), were transfected into two human cell lines (HMV-2/CVCL_1282 -Merck-, WM3211/CVCL_6797 -Rockland-) using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) following the manufacturer’s recommendations (Tables S1 and S3). Cells were seeded in 6-well plates in 1 mL of RPMI 1640 medium (Gibco, Amarillo, TX, USA), supplemented with 10% fetal bovine serum and 0.2% primocin (Invivogen, Waltham, MA, USA) at a density of 1 × 10⁶ cells, and incubated at 37 °C. The medium was changed 6 h post-transfection to avoid toxicity.
Cell proliferation was evaluated in 96-well plates, 72 h after transfection with an initial density of 30,000 cells per well, using a methylene blue colorimetric assay. Briefly, the cells were fixed for 30 min in 90% ethanol, removed, dried, and subsequently stained for 30 min using 1% methylene blue dye in borate buffer. The fixed cells were washed 4–5 times using tap water, and 100 µL of 0.1 N HCl was added to each well. Cell density was analyzed using a spectrophotometer at 620 nm.