Pcdh ef1 mcs t2a puro cd520a1

MDA-MB-231 and Hs578T breast cancer cells were cultured in DMEM with 10% FBS, 100 μg/ml streptomycin, 100 IU penicillin, and 1% L-glutamine at 37°C in 5% CO₂ and 95% air. For gene knockdown with siRNA, cells were transfected with 5 nM scramble siRNA or siRNA against Src kinase (6714, Dharmacon) for 72 h using lipofectamine RNAiMAX (Invitrogen, 13778-150). For lentiviral infection, HEK293T cells were seeded into 6-well plates then transfected with 2 μg pCDH-EF1-MCS-T2A-Puro (CD520A1, System Bioscience) encoding a luciferase expression vector, scramble shRNA or shRNA against Src (TRCN0000195339) using 6 μl lipofectamine 2000 (Invitrogen, 11668027) for 16 h. The transfected cells were then rinsed with media and incubated for 48 h. The supernatant was further used to infect MDA-MB-231 cells, which were maintained in culture with 1 μg puromycin added every 2–3 days to obtain stable cell lines.