Allstars cell death control sirna

For siRNA experiments, HEK293 cells were reverse transfected in 96-well format by incubating 2 pmol lyophilized siRNA in 50 μl OptiMEM with 0.2 μl Lipofectamine RNAiMax (ThermoFisher Scientific) for 30 min and then adding 5000 cells in 50 μl DMEM with 10% FBS, PS/Q. After 48 h, supernatants were removed and cells infected with 1000 TCID₅₀ rgEBOV-luc2 in a volume of 100 μl DMEM with 5% FBS, PS/Q. After an additional 48 h, 100 μl ONE-Glo reagent (Promega) was added to the cells, and reporter activity was measured after 10 min using a Glomax Multi microplate reader. Four biological replicates per siRNA in two independent experiments (two biological replicates per experiment) were obtained on a total of eight 96-well plates. On each plate, there were 8 wells each for negative siRNA (aneg #2), mock-infection, and no siRNA controls, and 4 wells each for the AllStars Cell Death Control siRNA (Qiagen) and the anti-EBOV L siRNA controls. For inhibitor experiments, HEK293 (EBOV, NDV, RABV) or VeroE6 (EBOV, IAV) cells were infected with GFP-expressing viruses at an MOI of 0.1 (EBOV, RABV, IAV) or 0.05 (NDV). Supernatants were harvested 48 h post infection, and titers were determined by TCID₅₀ analysis. To assess the effects on cell viability, a CellTiter-GLO assay (Promega) was performed in parallel following the manufacturer’s instructions.

Deregulation of miR-34a-3p expression in Ben-Men-1 cells was performed using miR-34a-3p (mimic) or anti-miR-34a-3p (inhibitor). AllStars Negative Control siRNA and miScript Inhibitor Negative Control were used as scrambled RNA negative controls for mimic and inhibitor, respectively. Transfections were performed using the lipid-based HiPerFect transfection reagent according to manufacturer's instructions. Transfection conditions were optimized using AllStars Cell Death Control siRNA (all from Qiagen, Hilden, Germany).