3.5. siRNA Transfer to MDA-MB-231 Cells

5.2 × 10⁴ or 10.5 × 10⁴ cells at high density were seeded in 48-well or 24-well plates, respectively, and incubated overnight. Before transfection the medium was replaced with serum-free one.Anti-GFP siRNA and mock siRNA-containing polyplexes were added in 48-well plates and incubated with cells for 2 and 4 h. L1 complexed with anti-AQP3, anti-CDC20, anti-COL4A2 and mock siRNAs were added in 24-well plates and incubated with cells for 2 h. The final concentrations of siRNAs were 100 or 200 nM and volumes of medium were 500 and 1000 µL for 48- and 24-well plates, accordingly. After incubation in full culture medium for the next 48 h, cells from 48-well plates were permeabilized for further GFP fluorescence measurement with a Wallac 1420D scanning multilabel counter (Thermo Fisher Scientific Oyj) at 485 nm excitation and 535 nm emission wavelengths as described previously [41]. The GFP fluorescence was normalized by the total protein concentration, measured with Bradford reagent (Helicon, Moscow, Russia). The results are presented as mean ± S.E.M from three independent experiments with three samples. After 48 h of incubation cells from 24-well plates were prepared for RNA extraction with Trizol reagent (Qiagen, Düsseldorf, Germany) and subsequent cDNA synthesis using an OT-1 Reverse Transcription kit (Syntol JSC) according to the manufacturer’s instructions.