Assay for PARPi and cisplatin resistance

MDA-MB-436 cells were seeded into six-well plates at a density of 1 × 10⁶ cells per well the day before transfection. Cells were transfected with 1 µg of empty vector or 2 µg of BRCA1 expression vectors using 6 µl of X-tremeGENE 9 (Roche). Cells were trypsinized 36 hr after transfection and reseeded into 10 cm plates at 100,000 cells per plate in a volume of 10 ml. Another 16 hr later, 1 ml of 10X G418 (6 mg/ml dissolved in the same culture medium) or 10X G418 containing 2.2 μM cisplatin or olaparib was added to each plate. The final concentrations of G418, cisplatin and olaparib were 550 µg/ml, 200 nM and 200 nM, respectively. All experiments were performed in duplicate plates for each construct. For cells selected with G418 alone, 15 days after selection, one plate was trypsinized and cells counted with a Vi-CELL Cell Counter (Beckman Coulter) to determine viable cell number, and the other plate was stained with Crystal Violet (0.5% w/v in 95% ethanol, 5% acetic acid) to count the number of colonies. For cells selected with both G418 and cisplatin, both plates were stained 21 days after selection. For cells subjected to G418 and olaparib double selection, both plates were stained 28 days after selection. Colonies with approximately 50 cells or more were counted, and the numbers were normalized against the number of viable cells on corresponding plates with G418 alone. The vast majority of constructs were measured by three or more independent experiments (each with duplicate sets of plates), with the only exceptions being when the first two experiments produced nearly identical results or were carried out using two independent plasmids.