Attractene transfection reagent kit

The plasmids of ShRNA snail1 containing reporter gene EGFP and the puromycin resistance cassette were designed and synthesized from Genecopoeia Inc (Rockville, MD, USA). The effective plasmid of ShRNA snail1 was filtered, which is listed in Table 3. Hep-2 cells were cultured in a 6 well plate until they reached 80% confluence; they were then transfected by ShRNA snail1 sequence for 24h with Attractene Transfection Reagent Kit according to the manufacturer's protocol (QIAGEN, Germany), and then selected by 0.4 μg/mL puromycin until stable clone formation. The transfection efficiency was determined by fluorescence intensity. The stable clones were dissociated and cultured in 1640 medium (Invitrogen, California, USA) containing 10% fetal serum and 0.2 μg/mL puromycin with 90% confluence for examinations.

To be able to visualize focal adhesion dynamics and quantify their lifetime, HaCaTs were transfected with mCherry-α-Paxillin-C1 construct kindly provided by Dr Irina Kaverina (Efimov et al., 2008 (link)). Transient transfection was performed following the Fast-Forward protocol provided by the Attractene transfection reagent kit (Qiagen, Germany). Briefly, 0.1 µg of the plasmid per sample was allowed to react with the Attractene reagent for 15 min at room temperature and then mixed with the cell suspension for cell seeding within the PDMS stencil placed on the hydrogel. After 12 hr, cell migration was initiated and mCherry signal was acquired every 2 min using an LSM 880 confocal microscope (Carl Zeiss, Germany) equipped with a 40× long-distance water immersion objective with a numerical aperture of 1.1. The obtained time-lapse videos were analysed using Imaris image analyses software (Bitplane, Oxford Instrument) automatically tracking each focal adhesion in the cells and quantified its lifetime area and length. The surface contact area of each cell was quantified by segmenting the mCherry signal in the cytoplasm at the focal plane where focal adhesions were visible. Focal adhesions density per cell was quantified by dividing the number of focal adhesions per the corresponding surface contact area.