Lentiviral packaging mix

To generate stable Notch3 knockdown (KD) cell lines using lentiviral vector, Notch3 shRNA and control lentiviral particles were generated in HEK293T cells by co-transfecting Notch3 shRNA or scrambled shRNA plasmids (Mission^® shRNA, Sigma-Aldrich, St. Louis, MO) and lentiviral packaging mix (Sigma-Aldrich) using Lipofectamine 2000 (Invitrogen, Waltham, MA) according to manufacturer’s instruction. Notch3 stable KD cell lines were achieved by infecting cells with lentiviral particles and followed by selection in puromycin-containing medium (1 μg/ml for 1205Lu; 2 μg/ml for A375 and WM852).

Lentiviruses were produced by co-transfection HEK293LTX cells with the MISSION® TRC2 pLKO.5-puro Non-Mammalian shRNA Control Plasmid DNA or with the pLKO-puro shRNA constructs (Sigma, shRNA sequences are available in Table S3) and the Lentiviral Packaging Mix (psPAX2 and pMD2.G). U2OS and NCIH1975 cells were transduced by the lentivirus, and cells with stable knockdowns were selected using puromycin (2 μg/ml for U2OS and 1 μg/ml for NCIH1975). The knockdown of RUNX1 and SNAI1 in stable cell lines was confirmed by quantitative real-time PCR.

HEK293T cell suspensions at 20,000 cells per well were dispensed into 96-well microtiter plates (Corning 3603) in 100 μL of media and incubated overnight at 37°C. The Lentiviral Packaging Mix (Sigma-Aldrich) containing the packaging plasmid and envelope plasmid were thawed from −20°C storage at room temperature in preparation for transfection. A master mix containing 16.5 μL of Opti-Mem (Life Technologies), 0.3 μL or 0.6 μL of FuGENE 6 and HD transfection reagent (Promega) diluted to 0.75 μL, and 1 μL of Lentiviral Packaging Mix was dispensed into an intermediate 96-well polypropylene storage plate (Corning 3357) containing 0.1 μg of expression plasmid and incubated for 15 minutes to promote plasmid-transfection reagent complex formation. Next, 20 μL was transferred into the cell plates and incubated for 48 h at 37°C. For the first harvest, 85 μL of lentiviral supernatant was transferred into an intermediate 96-well storage plate and stored at 4°C. Cell plates were replenished with 85 μL of media and incubated for an additional 24 h at 37°C. For the second harvest, 95 μL of lentiviral supernatant was transferred into the corresponding intermediate storage plate already yielding a total harvest of lentiviral particles in 180 μL. Plates were briefly centrifuged at 1,000 rpm to remove any cell debris and aliquots of lentiviral supernatants were stored at −80°C.