*2.4. Indirect Immunofluorescence Assay (IFA)*

Indirect immunofluorescence assay was used to rapidly evaluate antiviral activities of compounds against H5N1 IAV infection. For immunostaining, the H5N1-infected or control cells were fixed with 4% paraformaldehyde for 10 min, then permeabilized with 0.25% Triton X-100 for 10 min at room temperature (RT). Cells were blocked with 1% bovine serum albumin (BSA) for 60 min at RT and then incubated with a mouse monoclonal antibody against IAV nucleoprotein (NP protein) (1:500 dilution, Sino Biological, Beijing, China) at 4 ◦C overnight. After three washes with PBS, the cells were incubated for 1 h at RT with an anti-mouse IgG antibody conjugated with Alexa Fluor® 488 (green) (Cell Signaling Technology, MA, USA) at 1:1000 dilution. Nuclei were counterstained with 50 μL of 4,6-diamidino-2-phenylindole (DAPI, 300 nM; Sigma-Aldrich, MA, USA). Immunofluorescence was captured using the Leica DMI 4000B fluorescence microscope (Leica, Wetzlar, Germany). Blue and green fluorescence spots were counted as the total and IAV-infected cell numbers respectively in every IFA image.

Relative NP protein level (%) of each image was calculated based on the fluorescence optical density (OD) using Software Image J. Results from compound-treated samples were compared to those from corresponding DMSO-treated control groups (set as 100%). The EC50 value (the concentration required to protect 50% cells from IAV infection) was determined by plotting the relative NP protein level as a function of compound concentration and calculated using GraphPad Prism 7.0 software (GraphPad Software, San Diego, CA, USA).
