Apoptosis assay of COS-7 cells transfected with p3b/EGFP-N1. COS-7 cells were transfected with pEGFP-N1 and p3b/EGFP-N1 respectively. At 48 h after transfection, cells were collected and resuspended in binding buffer containing Annexin V-PE and 7-AAD, and then processed for flow cytometry analysis. On the left row, EGFP positive cells were gated. The middle and right rows were the results of EGFP negative and positive cells analyzed with Annexin V-PE and 7-AAD staining. In each box, the upper left corner included damaged cells, the lower left corner included viable cells, which were negative for 7-AAD and Annexin V-PE binding, the upper right corner included necrotic or late apoptotic cells, which were positive for Annexin V-PE staining and for 7-AAD uptake, while the lower right corner included apoptotic cells, which were Annexin V-PE positive but impermeable to 7-AAD. In p3b/EGFP-N1 transfected cells (a), the percentage of apoptosis cells in EGFP positive cells increased significantly, compared with negative ones, while there were no changes between positive and negative cells transfected with pEGFP-N1 (b). One of three experiments with similar results was shown.