siRNA-DNA co-transfection

HEK 293T cells were seeded at 1.2 × 10⁵ cells per well in 24-well culture plates. Endogenous Staufen1 protein silencing was performed over 70–80% confluent cells using the Lipofectamine 2000 system (#11668019, Invitrogen, ThermoFisher Scientific Inc). For Staufen1 silencing, a duplex siRNA 3084 (si55/63) (5′-AAATAGCACAGTTTGGAAACT-3′; Integrated DNA Technologies, IDT, Coralville, IA, USA) previously described (32), targeting Staufen1 was used at 50 nM for 48h, together with 200 ng of dl HIV-1 IRES or of the pNL-4.3-RLuc plasmid. For RLuc silencing, 100 nM of a duplex siRNA targeting the RLuc open reading frame (Renilla siRNA 2, 5′-UAUAAGAACCAUUACCAGAUUUGCCUG-3′, Integrated DNA Technologies, IDT) (24,61,62) was cotransfected with 150 ng of dl-plasmid and pSP64 Poly(A) or Stau1⁵⁵-HA₃ plasmid for 48 h. The Silencer Select Negative Control #1 siRNA (#4390844, Ambion, Thermo-Fisher Scientific Inc.), was used as a non-related scrambled RNA (scRNA) negative control.