Pcdh puro bcl xl

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)].

For PBMC culture, they were isolated using lymphoprep following the manufacturer´s instructions (Axis shield, Oslo, Norway) from septuagenarian individuals. After collecting and washing the mononuclear cells, 2×10⁶ cells were infected with a lentivirus containing Bcl-xL (pCDH-puro-Bcl-XL, Addgene, plasmid #46972) or the empty vector as previously described [46 (link)]. Cells were maintained in RPMI high glucose media supplemented with 10% FBS, 2 mM L-glutamine and penicillin (100 U/ml) and streptomycin (100 μg/ml)at 37°C in 5% CO₂ atmosphere,in the presence or absence of the mitogen PHA 1% (Sigma, St Louis, MO). Cell pellets were harvested for RNA analysis after 9 days in culture.

The short hairpin RNA (shRNA)-expressing lentiviral vectors (pSIH1-puro-STAT3 shRNA: #26596, pSIH1-puro-control shRNA: #26597) (Addgene, Watertown, MA, USA) and cDNA-expressing lentiviral vectors (pCDH-puro-BCL-X_L: #46972 [Addgene], pCDH-CMV-MCS-EF1-Puro: #CD510B-1 [System Biosciences, Palo Alto, CA, USA]) were used to stably express shRNA or cDNA, respectively. To generate lentiviruses, HEK 293T/17 cells were transfected with lentiviral vectors and packaging plasmids (Lentiviral High Titer Packaging Mix: Takara Bio, Shiga, Japan) using TransIT®-293 transfection reagent (Mirus Bio, Madison, WI, USA). On the following day, the medium was replaced with fresh growth medium, and lentivirus-containing supernatants were harvested and concentrated by centrifugation using a Lenti-X™ Concentrator (Takara Bio). To establish shRNA- and cDNA-expressing stable cell lines, the cells were transduced with lentiviral particles using 1 μg/mL Polybrene® (Santa Cruz Biotechnology, Dallas, TX, USA) and then selected with 2 μg/mL puromycin (Sigma-Aldrich) for 10–14 days. Stable gene knockdown was confirmed by western blotting.