Influenza Virus Infection and NXT1 Knockdown in MDCK and A549 Cells

MDCK (2.5 × 10⁵) or A549 (5 × 10⁵) cells were seeded in a six-well plate together with control or NXT1 siRNA (1.5 µL of 10 nM siRNA and 7.5 µL of RNAiMax in 500 µL of Opti-MEM) and cultured for 24 h or 48 h. The cells were infected with influenza A/WSN/33 virus at a multiplicity of infection (MOI) = 0.0001, as described previously [14 (link)]. The infected MDCK cells were cultured in DMEM supplemented with 10% FBS, with 1 µg/ml of tosylsulfonyl phenylalanyl chloromethyl ketone-treated trypsin (TPCK-trypsin; Worthington Biochemical Corporation, Lakewood, NJ, USA) to enhance viral replication efficiency while the infected A549 cells were cultured without TPCK-trypsin since the cells are sensitive to trypsin. Culture medium containing virus was collected at the indicated time points (see Figure 2) and titrated for virus concentration by plaque assay, as described previously [14 (link)]. In order to check viral protein expressions, the 24 h NXT1-knockdown MDCK cells at 18 h post-infection (hpi) and A549 cells at 48 hpi were collected, lysed with NET buffer (10 mM Tris-HCl/pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% NP-40) plus protease inhibitor cocktail, and boiled with SDS sample buffer for WB analysis of viral and cellular proteins (see Figure 2).