On targetplus non targeting pool

The following siRNAs were used: ON-TARGETplus Non-Targeting Pool (Thermo Scientific, D-001810–10), ON-TARGETplus Human MYC SMARTpool (Thermo Scientific, L-003282–02), ON-TARGETplus Human PAICS SMARTpool (Thermo Scientific, L-003980–00), siIMPDH2 #1: siIMPDH2 Silencer Select (Ambion, 106309), siIMPDH2 #2: ON-TARGETplus Human IMPDH2 SMARTpool (Thermo Scientific, L-004330–00) and Human p53 SMARTpool (Thermo Scientific, L-003329–00).

A549, MRC-5, and MDCK cells (all from the European Collection of Cell Cultures [ECACC]) and their derivatives were grown as monolayers in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C. Cycloheximide (CHX; used at 50 μg/ml), actinomycin D (ActD; used at 1 μg/ml), or 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB; 250 μM) was added to the medium at the time of infection, as indicated. Alpha-amanitin was used at 20 μg/ml and required a 1-h period of pretreatment before virus infection in order to efficiently inhibit viral protein synthesis (data not shown). Poly(I·C) transfections were carried out as described elsewhere (13 (link)). Small interfering RNA (siRNA) transfections were carried out using DharmaFECT (Thermo Scientific) according to the manufacturer's instructions. The sequences of NP-1496 and NP-231 siRNAs and their effects on influenza virus replication have been described previously (14 (link)) and were synthesized by Thermo Scientific. ON-TARGETplus Non-Targeting Pool (Thermo Scientific) was used as nontarget control siRNA.

U251vIII cells were seeded at a density of 30,000 cells/cm² and transfected with 75 nM human FPR2-targeting siRNA (Thermo, ID4150) or non-targeting control (NTC) siRNA (ON-TARGETplus non-targeting pool, Thermo) using lipofectamine 2000 according to the manufacturer’s protocol. The transfection was allowed to proceed for 20 h before seeding cells for Transwell cell migration assays.

Cells were transfected with 50 nmol/L of STAG2 siRNA (SMARTpool siGENOME siRNA, Thermo Fisher Scientific) or control siRNA (On TargetPlus nonTargeting pool, Thermo Fisher Scientific) using Lipofectamine 2000 (Invitrogen). Forty-eight hours after, a second transfection was performed with same conditions as the first transfection. The cells were cultured for another 72 hours before harvesting for Western blot analysis.

Oligonucleotides (desalted) were obtained from either Invitrogen or Sigma, whereas siRNAs (mouse anti-Gfap, anti-Vim, and anti-Lcn2 ON-TARGETplus SMARTpools, and the ON-TARGETplus Non-Targeting pool) were obtained from Thermo Scientific. All RNA solutions and buffers were prepared in RNase-free water.