Plasmids, plasmid transfection, and stable cell line generation

A mammalian expression plasmid that encoded human NNMT fused to Myc and DDK tags at its C terminus was obtained from Origene (Cat# RC200641). For the generation of stable NNMT overexpressing OVCAR-8 cell lines, the NNMT-Myc-DDK plasmid or empty vector control was transfected (5 μg/transfection) into OVCAR-8 cells (8 × 10⁶ cells/transfection) using a BTX ECM 830 electroporator (using a 4-mm cuvette with two, 280-volt, 10-msec pulses). Cells were plated in 10-cm dishes containing RPMI supplemented with 8% fetal bovine serum and incubated for 48 h. After G418 (2 mg/mL) was added, the cells were cultured for an additional 12 days, replenishing the selection medium every 3 days. G418-resistant clones were trypsinized using 0.25% Trypsin-EDTA (Life Technologies) and reseeded at 50 cells per dish into 15-cm dishes containing 2 mg/mL G418. After 10 days of culture, single colony clones were picked, expanded in 24-well plates containing complete medium plus G418. Seven to 10 days later, 10 empty vector or NNMT cell clones were isolated and assayed for NNMT by immunoblotting for each stably transfected cell line. To generate the SFB-BRCA1 mammalian expression plasmid, human full-length BRCA1 cDNA (15) was subcloned into the pSFB vector that contains in-frame N-terminal S-peptide, FLAG, and streptavidin-binding peptide tags (16). For the generation of stable BRCA1 overexpressing COV362 cells, the SFB-BRCA1 plasmid or empty vector control plasmid was transfected (40 μg/transfection) into COV362 cells (10 × 10⁶ cells/transfection), and after 48 h, the cells were cultured for an additional 21 days with G418 (1 mg/mL), replenishing the selection medium every 3 days. The G418-resistant cells were used for subsequent experiments.