Maintaining and Transfecting Cell Lines

A2780 human ovarian cancer cells (female) were maintained in RPMI-1640 medium (Sigma-Aldrich) supplemented with 10% (v/v) fetal calf serum, 1% (v/v) L-Glutamine and (v/v) 1% Antibiotic-antimycotic (both Sigma-Aldrich); telomerase-immortalized fibroblasts (TIF) cells, mouse-embryonic fibroblasts (MEF) cells and H1299 human lung cancer cells(male) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) containing L-Glutamine and supplemented with 10% (v/v) fetal calf serum, and (v/v) 1% Antibiotic-antimycotic (Sigma Aldrich); and N15A(Roberts et al., 2015 (link)) Pseudomyxoma peritonei (PMP) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma Aldrich) supplemented with 10% FCS, 25 mM HEPES, 5 μg/ml Insulin, 10 mM L-Glutamine and (v/v) 1% Antibiotic-antimycotic (Sigma Aldrich). All cell lines were incubated at 37°C in a humidified 5% (v/v) CO2 atmosphere. All siRNAs and fluorescent constructs were transiently transfected by electroporation using a nucleofector (Amaxa, Lonza) using solution T, program A-23, 3ug DNA / 5μl 20 mM siRNA as per the manufacturer’s instructions. Experiments were performed ∼24 h after nucleofection unless otherwise stated.