Figure 4. ERK signaling pathway participates in the hesperidin- and kaempferol- induced the opposite effects on influenza virus replication.

A549 cells were infected with influenza A/WSN/33 virus for 3 h, and then treated by ERK inhibitor (U0126) or hesperidin or kaempferol, respectively, or by combination of U0126 with hesperidin or kaempferol for 24 h, 0.05% DMSO as a control (PC). (A) Cells were stained by indirect immunofluorescence using NP or M1 specific rabbit antibody and FITC-coupled anti-rabbit IgG to determine cellular distribution and expression of NP and M1 proteins (Green), DAPI staining shows cell nucleus (Blue). (B) Statistically analysis of expression of NP and M1 proteins in nucleus using confocal laser scanning microscope FV1000 (OLYMPUS). (C) Statistically analysis of expression of NP and M1 proteins using flow cytometry (BD FACSCalibur). Data presented as means ± SEM of five visual fields for confocal and three independent experiments for FACS. H represented hesperidin; K represented kaempferol. (D) Virus growth curves. A549 cells were infected with influenza virus at a multiplicity of infection of 0.001 for multi-cycle replication and then treated with 100 μM of hesperidin or kaempferol or 0.05% DMSO, or with 10 μM of U0126 alone or 10 μM of U0126 combined with 100 μM hesperidin or kaempferol. At the indicated times, supernatants were collected, and then plaque titres were determined on MDCK cells. Each growth curve was represented by the average of three independent experiments.