Influenza A virus NS1 protein induces apoptosis in A549 cell. The transmission electron microscopy reveals that the cells transfected with pCMV-Myc empty vector shows an intact nuclear shape (A) and transfected with pCMV-Myc/3NS1 shows condensed, shrunk chromatins aggregated along inside the nuclear membrane, and apoptotic bodies (B). The flow cytometric analysis of A549 cells transfected with the empty pCMV-Myc vector (C) or transfected with pCMV-Myc/3NS1 (D). The dot plot diagrams represent typical apoptotic and necrotic cell populations detected by Annexin V-FITC and PI staining. The lower left quadrants of the panels show viable intact cells, which were negative for Annexin V-FITC binding and excluded PI staining (FITC-/PI-); the upper right quadrants show nonviable, necrotic cells, which were positive for Annexin V-FITC binding and PI uptake (FITC+/PI+). The lower right quadrants represent apoptotic cells, positive for Annexin V-FITC and negative for PI (FITC+/PI-). The expression of caspase 3 and caspase 9 posttransfection 0 h, 12 h, 24 h, and 48 h were detected by Western blotting (E), and the enzyme activities of caspase-3 and caspase-9 in the NS1-transfected A549 cells were detected by Caspase-3/CPP32 Colorimetric Protease Assay and Caspase-9/Mch6 Colorimetric Assay, respectively (F).