NEDD4 knockdown in human lung cells increases IFITM3 levels and resistance to influenza virus infection.

<p>A-C) A549 cells were transfected for 48 h with control siRNA (siControl) or siRNA targeting human NEDD4 (siNEDD4). A) Cells were collected just prior to infection for confirmation of NEDD4 knockdown by anti-NEDD4 Western blotting, with anti-actin blotting serving as a loading control. Anti-IFITM3 blotting demonstrates an increase in IFITM3 upon NEDD4 knockdown. B,C) Following siRNA treatment, cells were infected with influenza virus strain PR8 at an MOI of 2.5 for 6 h. Cells were then fixed and stained with anti-influenza virus NP to measure the percentage of cells infected using flow cytometry. Results shown are representative of three independent experiments, with samples run in triplicate. Error bars represent standard deviation of triplicate samples. * Indicates a p-value less than 0.0001 calculated with Student’s t-test. D) NCI-H358 and NCI-H2009 cells were transfected for 48 h with siControl or siNEDD4. Cell lysates were subjected to immunoblotting with anti-NEDD4 to confirm NEDD4 knockdown, anti-IFITM3 to demonstrate increase in endogenous IFITM3 upon NEDD4 knockdown, and anti-GAPDH as a loading control. E) A549 cells were treated with equal volumes Dimethyl Sulfoxide (DMSO) as a control, MG132 (10 μM), Chloroquine (10 μM), Bafilomycin A1 (1 μM), or Leupeptin (100 μM) for 24 h. Cells were also treated with IFN-α (100 units/mL) for comparison. Cell lysates were subjected to anti-IFITM3 immunoblotting to evaluate endogenous IFITM3 levels with each treatment. Western blotting with anti-GAPDH served as a loading control.