Evaluating IAV NS1 Effect on Protein Synthesis

To evaluate the effect of IAV NS1 on host protein synthesis, confluent monolayers of human 293T cells (96-well plate format; 6 × 10⁴ cells/well in triplicate) were transiently cotransfected, using DNA-IN, with 200 ng/well of pCAGGS-HA NH2 NS1 expression plasmids, or the empty plasmid as a control, together with 50 ng/well of pCAGGS plasmids expressing GFP (17 (link)) and Gaussia luciferase (Gluc) (34 (link)). GFP and Gluc expression levels were measured 30 hpt using fluorescence microscopy and a Lumicount luminometer, respectively. For Gluc measurements, cell culture supernatants were mixed with an equal volume of Biolux Gaussia luciferase reagent (New England BioLabs).