Due to the type of chemical modification, only the antisense strand can participate in RNAi, thus avoiding not only unwanted, sense strand-mediated, off-target effects but also preventing any possible interference of the sense strand with adenoviral transcripts generated from the opposite viral DNA strand not intended to be targeted. Besides, this type

of modification (frequently present in similar versions ATM/ATR inhibitor drugs in commercial siRNAs) can increase the intracellular half-life of siRNAs and reduce their cytotoxicity. The pTP-si1 to pTP-si4 siRNAs (obtained from Ambion/LifeTechnologies Austria, Vienna, Austria) were 21-mer, unmodified siRNAs carrying two nucleotide (nt) TT overhangs at their 3′ ends and were also included in our experiments. As negative controls, two distinct universal non-targeting siRNAs (Invitrogen, Ambion), matching the type of design of the respective targeting siRNAs, were employed. SiRNAs were designed using the Invitrogen BLOCK-iT™ RNAi Designer or Dharmacon Raf inhibitor siDESIGN tools

and target site accessibility, as calculated by RNAxs (http://rna.tbi.univie.ac.at/cgi-bin/RNAxs), was taken into account. 1.4e+05 HEK293 and 3e+04 A549 cells were seeded into the wells of 96-well plates, and reverse transfected with 50 ng of individual dual-luciferase reporter vectors and 30 nM targeting or non-targeting control siRNA using Lipofectamine 2000 (Invitrogen/LifeTechnologies Austria, Vienna, Austria). Briefly, for each well 0.5 μL Lipofectamine 2000 was diluted with 24.5 μL OptiMEM medium (Invitrogen/LifeTechnologies Austria, Vienna, Austria), and after 5 min of incubation, 25 μL diluted Lipofectamine 2000 was mixed with 25 μL of a specific siRNA/reporter vector mix (diluted in OptiMEM). After 20 min of incubation, the mixes were pipetted directly into the wells of a 96-well plate

and freshly harvested cells were added. After 24 h of incubation, OSBPL9 medium was exchanged and cells were incubated for another 24 h. Culture conditions were as described above. Firefly and Renilla luciferase activities were determined at 48 h post-transfection using the Dual-Glo luciferase assay (Promega), according to the manufacturer’s instructions. Briefly, 75 μL of Dual-Glo Reagent was added to cells grown in 75 μL medium, and after 10 min of incubation at room temperature, firefly luciferase activity was measured. Next, one volume of Dual-Glo Stop & Glo reagent was added to each well, plates were incubated for an additional 10 min at room temperature, and eventually, Renilla luciferase activity was determined. Luminescence was measured on a Wallac Victor 1420 Multilabel Counter (Perkin Elmer Austria, Brunn am Gebirge, Austria). Knockdown rates were calculated by normalizing Renilla luciferase activities to firefly luciferase activities, and comparing dual-luciferase ratios between targeting and non-targeting control siRNAs. 1.