Proliferation and apoptotic cell death assessment
Proliferation, apoptosis and RNA expression were investigated 48h post- transfection. Proliferation was assessed by colorimetric detection based on 5-bromo-2’-deoxyuridine (BrdU) incorporation (Roche, Mannheim, Germany), according to manufacturer’s protocol. Briefly, cells were incubated with BrdU for 3h, and absorbance was read at 370nm (SpectraMax Plus 384 Microplate Reader, Molecular Devices LLC, San Jose, CA, USA) 48h post- transfection. Cells incubated for few seconds with BrdU- were used as background control.
Apoptosis was measured 48h post- transfection by photometric detection of cytoplasmic histone-associated-DNA-fragments in cell lysates according to manufacturer’s instructions (Roche). Briefly, cell lysates were loaded on to a streptavidin-coated plate and subsequently incubated with biotinylated anti-histone antibodies and anti-DNA antibodies conjugated with peroxidase (POD). Unbound antibodies were removed by washing. Substrate solution, tetramethyl-benzidine (TMB) (Roche), was added. The peroxidase activity in the immunocomplex was determined photometrically at 405nm.