Inhibition of influenza viral NP protein and RNA synthesis by BPR1P0034. MDCK cells were infected with influenza virus A/WSN/33 (MOI = 0.5) and incubated with or without 5 μM BPR1P0034 in the adsorption and postinfection stages. The sample was subjected to western blotting (A), immunofluorescence microscopy (B), or quantitative PCR (C). (A) The infected cells were harvested at the times indicated and evaluated by western blotting using an anti-NP antibody. GAPDH was used as the loading control. This is representative of two independent experiments. (B) Influenza A/WSN/33 virus (MOI = 5)-infected MDCK cells on coverslips were incubated with or without BPR1P0034 during viral adsorption and after infection. The cells were fixed for the indicated times and reacted with primary anti-NP antibody and Alexa-Fluor-488-labeled secondary antibody. The production of viral protein, represented by NP, was detected by immunofluorescence microscopy. (Row A) Cells infected with influenza virus and harvested at 3 h p.i; (Row B) The same as (Row A) but in the presence of BPR1P0034; (Row C) Cells infected cells and harvested at 6 h p.i; (Row D) The same as (Row C) but in the presence of BPR1P0034. (C) The MDCK cells were challenged with virus in the presence or absence of BPR1P0034 and total RNA was extracted at indicated times. Equal amounts of total RNA (3 μg) were used for quantitative RT-PCR analysis. To quantify the changes in gene expression, the ΔCt method was used to calculate relative changes which were normalized to the GAPDH gene. The ratio of viral RNA to the internal control was normalized to the RNA level at 0 h p.i, which was arbitrarily set to 1.