Fig. 2

Effects of 5-MF on IAV-triggered apoptosis. A Immunofluorescence four-color staining showed colocalization of CD8⁺ (green), granzyme B (yellow), and TNF-α (red) in the lungs. Nuclei were stained with DAPI (blue). B The fluorescence intensities for granzyme B and TNF-α in CD8⁺ T cells were quantified. C Determination of apoptotic (green, TUNEL-positive; red, active caspase 3) lung epithelial cells (yellow, EpCAM) in the lungs by four-color immunofluorescence staining. D The fluorescence intensities for TUNEL and active caspase 3 were quantified. E Determination of the levels of Bax (yellow) and Bcl2 (red) in lung epithelial cells (green, EpCAM) in the lungs by four-color immunofluorescence staining. F The fluorescence intensities for Bax and Bcl2 were quantified. G The cytotoxicity of 5-MF (0–200 μM) on A549 cells was measured using the MTT assay. H H1N1 virus-infected A549 cells with 5-MF (0–30 μM) incubation for 24 h, and cell apoptosis in each group was detected by flow cytometry. I The percentage of apoptosis in H1N1 virus-infected A549 cells with or without 5-MF treatment. J Levels of TRAIL in H1N1 virus-infected A549 cells with or without 5-MF incubation were measured by Luminex assay. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 relative to the control group; ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 relative to the virus group