For stable overexpression of SNAI1, plasmid encoding full length wide type SNAI1 cloned from pCMV-Entry-SNAI1 (Origene) into pLenti-GIII-CMV-GFP-2A-Puro vector (ABM). Empty vector with no inserts was used as negative control. Plasmids were mixed with Mission Lentiviral Packaging Mix (Sigma-Aldrich) before added to a mixture of transfection reagent Fugene 6 (Roche). After 15 minutes incubation at room temperature, plasmid mix was added to 293T cells and viral supernatants were harvested at 48 and 72 hours post-transfection. Cells infected with lentivirus were selected using puromycin at a proper concentration decided by their respective puromycin kill curve.
To generate Tet-inducible SNAI1 expressing cells, SNAI1 was cloned from pCMV6-Entry-SNAI1 vector using into pLVX-TRE3G vector (Clontech). pLVX-TRE3G-SNAI1 was cotransfected with pLVX-EF1Alpha-Tet3G (Clontech) to 293T cells to generate lentiviral supernatants. Cells infected with lentivirus were dually selected using puromycin and G418. Same primer pairs containing BamHI and EcoRI were used for generating both vectors and were listed in Supplementary TableS1 .
To generate Tet-inducible SNAI1 expressing cells, SNAI1 was cloned from pCMV6-Entry-SNAI1 vector using into pLVX-TRE3G vector (Clontech). pLVX-TRE3G-SNAI1 was cotransfected with pLVX-EF1Alpha-Tet3G (Clontech) to 293T cells to generate lentiviral supernatants. Cells infected with lentivirus were dually selected using puromycin and G418. Same primer pairs containing BamHI and EcoRI were used for generating both vectors and were listed in Supplementary Table