*2.5. Cell Transfection*

For standard transfection experiments, the cells were seeded at a density of 35,000 cells per well of a 24 well plate in 0.5 mL fully supplemented medium. The next day, the cells were transfected with polyplexes containing 30 pmol/0.4 μg siRNA per 24 well as described above in the presence of FCS and without further medium change. For siRNA targets and sequences see Table S1.

#### *2.6. Determination of Knockdown E*ffi*cacies and Complex Uptake*

Luciferase activities were determined 72 h after transfection using the Beetle-Juice Kit (PJK, Kleinblittersdorf, Germany). The medium was aspirated and the cells were lysed with 300 μL Luciferase Cell Culture Lysis Reagent (Promega, Mannheim, Germany) for 30 min at RT. In a test tube, 10 μL lysate was mixed with 25 μL substrate and the luminescence was immediately measured in a luminometer (Berthold, Bad Wildbad, Germany).

The EGFP expression was determined by flow cytometry after 72 h. The cells were trypsinized, centrifuged for 3 min at 3000 rpm, and resuspended in 0.5 mL PBS, 1% BSA. The cells were measured in an Attune® Acoustic Focusing Cytometer (Applied Biosystems, Foster City, CA, USA). Finally, 20,000 events in the vital gate were analyzed using the Attune® software (V2.1.0).

For uptake experiments, complexes were prepared as described above (Section 2.4), but using a fluorophore-labeled siRNA-Atto488. PC3 wildtype cells were transfected, after 24 h prepared as described above with an additional PBS washing step and analyzed by flow cytometry.

To visualize the complex uptake by confocal microscopy, PC3 wildtype cells were seeded at a density of 200,000 cells onto glass coverslips in a 6 well plate. The next day, the cells were transfected with complexes of the different tyrosine-modified PEIs containing Alexa Fluor ® 647-labeled siRNA (2 μg siRNA/well). After 36 h, the siRNA uptake and intracellular distribution was analyzed by confocal microscopy (TCS SP8, Leica, Wetzlar, Germany).

#### *2.7. Cell Proliferation and Viability Assays*

For proliferation assays, cells were seeded at a density of 2000 cells (PC3 and Saos-2) or 1000 cells (MV3) per well of a 96 well plate in 100 μL medium on the day before transfection. For endpoint cell viability assays, PC3 or G55T2 cells were seeded at a density of 7000 cells per 96 well in 100 μL medium. The number of metabolically active cells after transfection was determined by using a colorimetric assay (WST-8 Cell Counting Kit-8; Dojindo Molecular Technologies EU, Munich, Germany). Briefly, after aspirating the medium, 50 μL of a 1:10 dilution of WST-8 in serum-free medium was added to the cells and incubated at 37 ◦C for 1 h. The absorbance was measured at 450/620 nm in a plate reader.

Acute cell damage caused by the polyplexes was determined by measuring the extent of lactate dehydrogenase (LDH) release, using the Cytotoxicity Detection Kit (Roche, Mannheim, Germany) according to the manufacturer's manual. Briefly, conditioned medium from transfection experiments (negative control siRNA) was collected after 24 h. For the determination of the maximum LDH release, cells were lysed with Triton X-100 at a final concentration of 2%, and conditioned medium from untreated cells served as negative control. In a 96 well plate, 50 μL sample medium was mixed with 50 μL reagent mix and incubated for 30 min in the dark. The reaction was stopped with 50 μL 1 M acetic acid and the absorption at 490/620 nm was measured using a plate reader. For blank value correction, fresh fully supplemented medium was mixed with the reagent and subtracted from all values.
