Transfection of plasmid DNA

Target cells were seeded one day prior to transfection to achieve 90–100% confluency at the time point of transfection. After a medium change 30 min before transfection, cells were transfected using the Trans-IT-LT1 transfection reagent (Mirus, Madison, WI, USA), according to the protocol of the manufacturer, except for 10 cm-diameter culture dishes where 10 μg of DNA, 30 μl of Trans-IT-LT1 reagent and 800 μl transfection medium were used. Reduced serum Opti-MEM medium (ThermoFisher Scientific) was used for preparing transfection complexes. In the case of co-transfection of two constructs, equal amounts of each plasmid DNA were used to reach a total DNA amount required for the given format. In the case of EM or immunofluorescence analysis, medium was changed 4 h post transfection. Cells were lysed or fixed 16 to 20 h after transfection and processed for subsequent assays.