Fig. 6

AMPKα was involved in the antiviral action of 5-MF. A Western blotting was performed to detect the expression of P-AMPKα, P-P53, and P53 in H1N1 virus-infected A549 cells. B The relative protein band intensities of P-AMPKα, P-P53, and P53 were quantitated using ImageJ software. C Immunofluorescence analysis was performed to detect the phosphorylation of AMPKα in the lung tissues. D The fluorescence intensity for P-AMPK was quantified. E H1N1 virus-infected A549 cells were pretreated with compound C (10 μM) for 30 min, followed by 5-MF treatment for 24 h. Western blotting was performed to analyze the expression of RSAD2. F The relative protein band intensity of RSAD2 was quantitated using ImageJ software. G The culture supernatants were collected from H1N1 virus-infected A549 cells with or without 5-MF 24 h treatment, and then transferred to uninfected A549 cells for 15 min of stimulation. Western blotting was performed to analyze the expression of P-JAK1, P-STAT1, and P-STAT2. H ELISA assay was performed to measure the levels of IFN-β in the culture supernatants. I A549 cells were infected with H1N1 viruses for 4 h, and then stimulated with recombinant human IFN-β (20 ng/mL) for 15 min. Western blotting was performed to analyze the expression of P-JAK1, P-STAT1, and P-STAT2. J After pretreatment with 5-MF alone or in combination with compound C (10 μM) for 4 h, MDCK cells were infected with H1N1 viruses for 1 h. Cells were stained with acridine orange (4 μg/mL) for 15 min and subsequently analyzed by confocal microscopy. ^#p < 0.05 relative to the control group; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 relative to the virus group; ^δδδp < 0.001 relative to the H1N1 virus + 5-MF (20 μM) group; ^ξξξp < 0.001 relative to the H1N1 virus + 5-MF (30 μM) group