siRNA-mediated knockdown of ExoRDec genes in A549 cells to study influenza virus replication

siRNAs were purchased from Dharmacon (ON-TARGETplus SMARTpools and Non-Target Control pool). Individual siRNAs targeting ERI1 were purchased from Sigma Aldrich. A549 cells were transfected with 25 nM siRNA with the Interferine transfection reagent (Polyplus). siRNAs targeting FANCG, COPS5 and NUP62 factors, known from the literature to be required for IAV replication, were used as controls (43 (link),44 (link)). At 48 h post-transfection, cells were infected with the A/WSN/33(H1N1) at a multiplicity of infection (moi) of 10⁻⁴ pfu/cell or with A549 cell-adapted A/Bretagne/7608/2009 (H1N1pdm09) and A/Centre/1003/2012(H3N2), or with A/Paris/650/2004(H1N1) (moi 10⁻³ pfu/cell) virus for 24 h. Plaque assays with MDCK-SIAT cells were performed as described in (45 (link)). Cell viability was determined using the CellTiter-Glo Luminescent Viability Assay kit according to the manufacter's instructions (Promega). To control the efficiency of siRNAs targeting ExoRDec genes, siRNA-treated A549 cells were transfected with plasmids encoding ExoRDec proteins fused with the full-length Gaussia luciferase (pGlucFL-ExoN) using linear PEI (polyethylenimine). The luciferase activity was measured 24h later in cell lysates using the Renilla luciferase assay reagent (Promega) and a Berthold CentroXS luminometer as described before for GPCA.