Lenti x 293t

The viral packaging cell line Lenti-X 293T was obtained from Clontech and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (CellGro) and 10% FBS (HyClone). To generate lentiviruses, the Lenti-X 293T was transfected with 20 µg psPAX2 and 6 µg pMD2.G packaging plasmids (Addgene), and 15 µg viral plasmid using Lipofectamine 2000 reagent (Life Technologies) on a p100 plate. Harvesting the virus was performed at 48 and 72 h after transfection and the collected media was pooled. The virus was concentrated using Lenti-X Concentrator (Clontech) and resuspended in 1 mL of serum-free PBS to increase its concentration ten-fold. PC9 cells and HCC827 cells were seeded in PS-free growth media on six-well plates at 5 × 10⁵ per well and 1 × 10⁶ per well, respectively. The cells were transduced next day with lentiviruses overnight. In total, 30 min before transduction, media was changed for media containing 6 µg/µL polybrene (Sigma). Cells were treated with 25 µL of a 10x virus stock. Cells were washed twice from the viruses with PBS and recovered for 8–10 h in regular growth medium before splitting in a 96-well plate at density of 8 × 10³ cells/well or 2.1 × 10⁴ cells/well for PC9 and HCC827 cell lines, respectively. Erlotinib treatment was initiated next morning.

Overexpression of Bcl-2 and Bcl-xL in MCF10A cells was performed by lentiviral transduction using the Lenti-X Packaging System (Clontech, Mountain View, CA, USA) according to the manufacturer’s instructions. The pCDH-puro-Bcl2 plasmid (Addgene plasmid #46971) and pCDH-puro-Bcl-xL (Addgene plasmid #46972) were added into supplied nanoparticle complexes for 10 min and applied to Lenti-X 293T cells to produce the virus. The medium was changed after 24 h and viral supernatant was harvested after 48 h, filtered, and used to infect cells at an approximate MOI of 10 along with 1 µg/mL Polybrene. The plate was then immediately centrifuged for 1.5 h at 1000 rpm. Cells were selected with 1 µg/mL puromycin at 2 days after infections and maintained in puromycin-containing media. Puromycin was removed from the medium for experimental conditions. pCDH-puro-Bcl2 and pCDH-puro-Bcl-xL were gifts from Jialiang Wang [36 (link)].