Allstars hs cell death

Two small interfering RNA (siRNA) molecules were used against ESPL1 (Hs_ESPL1_5, 5′-CAGCAGCTGACTGCTAAGCTA-3′; Hs_ESPL1_6; 5′-TACCTCCAAGGTTAGATTTAA-3′) (Qiagen, Hilden, Germany) to generate transient silencing of this gene. Each siRNA at a final concentration of 5 nM was added to individual wells in a 6-well plate and complexed with lipofectamine RNAiMAX (ThermoFisher Scientific, Waltham, MA, USA) in serum-free medium for 30 min. Cells were then added in medium supplemented with 20% FBS to yield transfection mixtures in media containing 10% FBS. Final transfection mixtures were incubated at room temperature (RT) for 1 h before being placed at 37 °C in a humidified atmosphere containing 5% CO₂. AllStars Hs Cell Death (Qiagen) was used as a positive cell death phenotype control, and AllStars Negative Control (Qiagen) was used as a negative control. Target-specific transfection efficiency was confirmed after 72 h at the mRNA level by real time RT-qPCR and after 96 h at the protein level by Western blot analysis. For both evaluations, mRNA and protein levels were compared with those found in cells transfected with negative control siRNA. Functional studies were conducted at 72 h post-transfection, except for cell viability analysis, which was conducted in 96-well plates at 96 h post-transfection and all reagent amounts were scaled down 30-fold.