293tn cells

The GeneNet h50K siRNA library from SBI was packaged according to the manufacturer’s recommendations. Briefly, ten 15cm plates were seeded at 7 × 10⁶ 293TN cells (SBI, Palo Alto, CA) per plate, resulting in a confluency of 60–80% 24 hr later. Each plate was transfected with 5 μg of the pooled library plasmids and the pPACKH1 packaging plasmids (22.5 μg) (SBI, Palo Alto, CA) using Lipofectamine (Thermo Fisher Scientific, Waltham, MA). 48 hr after transfection, the supernatant containing virus was harvested, clarified and concentrated using PEG-it (SBI, Palo Alto, CA) at ratio of one-part PEG-it to five parts supernatant overnight at 4°C. The supernatant + PEG-it solution was centrifuged for 20 minutes at 4°C and the pelleted virus + PEG-it was resuspended in 2.4 ml of PBS. Virus was stored at −80°C in 25 μl aliquots. The viral preparation was titrated using the Global UltraRapid Lentiviral Titering Kit (SBI, Palo Alto, CA) according the protocol. The optimal MOI of 0.51 to achieve a transduction efficiency of ~40% in the target cells (less than one copy per cell) was determined by flow cytometry using a GFP-expressing viral construct with the same backbone as the shRNA library.

We purchased MDA-MB-231 and MCF7 breast cancer cell lines from ATCC. The MDA-MB231BrM-2a, CN34 and CN34-BrM2c cells were a kind gift from Dr. Joan Massagué (Memorial Sloan-Kettering Cancer Center)^{37 (link)}. The MDA-MB-231-HM cells were a kind gift from Shizhen Emily Wang (City of hope)^{17 (link)}. Firefly luciferase-labeled cells were generated by lentivirus infection. 293TN cells were obtained from System Bioscience. MDA231, MDA231 variant cells and 293TN cells were cultured in DMEM medium supplemented with 10% FBS and antibiotics. CN34 and its variant cells were cultured in Medium199 supplemented with 2.5% FBS, 10 µg/ml insulin, 0.5 µg/ml hydrocortisone, 20 ng/ml EGF, 100 ng/ml cholera toxin and antibiotics. The immortalized mouse brain microvascular endothelial cells (mBrEC) were supplied by Dr. Isaiah J. Fidler (M.D. Anderson Cancer Center)^{38 (link)}. mBrEC was maintained at 8% CO₂ at 33 °C in DMEM with 10% FBS, 2 mM of L-glutamine, 1 mM of sodium pyruvate, 1% of non-essential amino acids and 1% of vitamin mixture. Primary astrocytes were purchased from clonexpress.

Pseudoviral particles were produced as described previously [68 (link)]. In brief, 293-TN cells (System Biosciences, SBI) were co-transfected with the lentiviral plasmid and lentiviral helper plasmids (psPAX2 and pMD2.G, gifts from Didier Trono) using PureFection (SBI), following manufactures protocol. Retroviral particles were produced using 293-GPG cells, which contain the viral packaging plasmid inside. 293-GPG cells were also transfected with plasmid of interest using PureFection. Lentivirus or retrovirus containing supernatants were collected at 48 hours and 72 hours after transfection, and filtered through a 0.45 μm filter. Cells were seeded into 6-well plates so that they reached 50–60% confluency on the day of infection and transduced at most 3 consecutive days with the viral stock in the presence or absence (for BT-549 cell line) of 8 μg/mL polybrene (Sigma-Aldrich). The viral solution was replaced with fresh culture medium, and cells were cultured for 24 hours before selection with 1 μg/mL of puromycin (InvivoGen) or 6ug/mL of blasticidine S (InvivoGen) for 10 days. Cells were also sorted using fluorescence assisted cell sorting (FACS) to ensure expression of all the fluorescent reporters and maintained in a media with antibiotics to ensure continued plasmid expression.