Lenti pac hiv mix

HEK 293Ta cells were cotransfected with 2.5 µg lentiviral C-Myb (GeneCopoeia, USA) plasmids, 5 µL Lenti-Pac HIV mix, and 15 µL EndoFectinLenti following the manufacturer's protocols (GeneCopoeia, USA). After 48 h of transfection, particles were collected from cell medium by 10 min centrifugation at 500 g and 0.44 µm filtration. To increase the transfection efficiency, virus-containing filtrate, mixed with isometric serum-free medium containing 20 µg/mL polybrene (Sigma, USA), was added to cells overnight and replaced with regular culture medium for subsequent 24 h incubation. Finally, cells used for follow-up experiments were selected by 10 µg/mL puromycin (Sigma, USA) for 48 h.

Lentivirus-expressing IRF6 shRNA were produced to structure stable IRF6 knockdown cell lines. Two more effective shRNA sequences (shRNA plasmid DNAs purchased from Obio Technology; the TRC numbers for the IRF6 sh1 and sh2 plasmids are Y3667 and Y3668, respectively) were selected for the following research. Lentiviruses were generated by 293T cells with the shRNA using the X-tremeGENE HP DNA transfection reagent (Roche). Forty-eight hours after transfection, the harvested infectious lentiviruses were filtered through a 0.45 µm filter (Millipore, GLQ025), transduced with lentiviruses IRF6 shRNA and control shRNA, and selected with 2 μg/mL puromycin (SIGMA, A1113803) for 5–7 days. The efficiency of the IRF6 knockdown was analyzed using real-time PCR and immunoblotting.
Lentiviral plasmids were co-transfected with Lenti-pac HIV mix (Genecopoeia, LT003) and EndoFection mix (Genecopoeia, LT003) into 293T cells to establish the IRF6 stable overexpression MDA-MB-231 cell line. A homologous vector carrying a Flag sequence was used as the control. Cells were transfected with lentiviruses IRF6 or vector. Stable cell lines were selected in 2 μg/mL puromycin for 5–7 days and the IRF6 overexpression efficiency was confirmed by RT-qPCR and immunoblotting.