The AlamarBlue (AB) reagent (Life Technologies, Carlsbad, CA) was used to assess the effect of test compounds on cell viability. Seeded A549 cells were treated with compounds using a 10-point twofold serial dilution (50 to 0.1 µM) and incubated for 48 h. AB was added to a final concentration of 10% (v/v) and incubated for 4 h and fluorescence measured at an excitation/emission of 545/590 nm. The percentage reduction in AB was calculated using the following controls: 0% reduced (DMEM + AB) and 100% reduced (Cells + DMEM + AB).
To assess compound effect on global cellular protein synthesis, cells were treated with compound for 24 and 48 h prior to labeling with [35S]Met/Cys pro-mix (PerkinElmer) for 1 h. To determine the effect of compound on SeV replication, cells were treated for 2 h followed by infection for 18 h prior to labeling as above. Whole-cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by isotope incorporation visualization and quantification using a FLA-5000 phosphoimager (FujiFilm, Tokyo, Japan) and Image Studio software (Li-Cor, Lincoln, NE).
To assess compound effect on global cellular protein synthesis, cells were treated with compound for 24 and 48 h prior to labeling with [35S]Met/Cys pro-mix (PerkinElmer) for 1 h. To determine the effect of compound on SeV replication, cells were treated for 2 h followed by infection for 18 h prior to labeling as above. Whole-cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by isotope incorporation visualization and quantification using a FLA-5000 phosphoimager (FujiFilm, Tokyo, Japan) and Image Studio software (Li-Cor, Lincoln, NE).