Plasmid transfection

A549 or Beas2B cells were subcultured on 6-well plates, 35-mm plates, or 100-mm dishes to 70%–90% confluence. Lipofectamine 2000 transfection reagent was added to the mixture containing varying amounts of plasmid and 200 μl of Opti-medium, and then incubated for 10 min to allow transfection reagent/DNA complexes to form. The mixture was added directly to the cells with complete medium. MLE12 cells grown on 100-mm plates (70%–90% confluence) were transfected with plasmids using Lonza electroporation transfection according to the manufacturer’s protocol, and then cultured for 48 h. Protein transient expression were confirmed by western blotting.