Sikras esirna

For cell viability assessment via MTS assay, 3000 A549 cells were seeded per well in phenol red free RPMI-1640 medium (Sigma-Aldrich) in a 96-well plate 24 h prior to the experiment. Cells were transfected with 100, 150 and 200 μg of empty BSA nanoparticles and incubated for 48 h or with 200 μg of BSA nanoparticles for 24, 48, 72 and 96 h at 37°C, 5%CO₂. Untreated cells were correspondingly incubated as blank controls. For assessment of cell viability after siRNA mediated knock down, 5000 A549 cells were seeded per well in phenol red free RPMI-1640 medium in a 96 well plate. Cells were then treated with non-specific, scrambled siCtrl, siKRAS (esiRNA, Sigma Aldrich) or siKRAS G12S (Eurofins Genomics, Ebersberg, Germany) at a final siRNA concentration of 30 nM. Cells were incubated for 48 h at 37°C, 5% CO₂. Subsequently, 20 μl of CellTiter 96® AQueous One Solution (Promega, Madison, USA) was added to each well and incubated for 4 h at 37°C, 5% CO₂ before absorption measurements were performed at 490 nm using a FLUOstar Omega (BMG Labtech, Ortenberg, Germany).

For apoptosis assessment via Annexin V and Propidium iodide staining, 50,000 A549 cells were seeded per well in a 24 well plate in RPMI-1640 complete medium 24 h prior to the experiment. Cells were then treated with non-specific, scrambled siCtrl, siKRAS (esiRNA, Sigma Aldrich) or siKRAS G12S (Eurofins Genomics, Ebersberg, Germany) at a final siRNA concentration of 30 nM. Cells were incubated for 48 h at 37°C, 5% CO₂. Subsequently, cells were washed two times in cold PBS and resuspended in Annexin V Binding Buffer at a concentration of 1 × 10⁶ cells/ml, and 100 μl of cell suspension was taken from the suspension above to be treated with 5 μl of Annexin V-FITC (BD Biosciences) and 10 μl Propidium iodide (BD Biosciences). The cell suspension was incubated for 15 min in the dark, following which 400 μl of Annexin V Binding buffer was added to each tube before measurement using the Attune™ NxT Acoustic Focusing Cytometer.