Fig. 3

EOAI induces intrinsic apoptosis. A A549 and H460 cells were treated with EOAI for 48 h. Annexin V-FITC/PI double-staining determined apoptosis. Representative FACS pictures are shown in the left panel. The cells in the second (upper right) and fourth (lower right) quadrants of the scatter plots were Annexin V + . The percentage of Annexin V + cells was defined as apoptosis rate and shown in the right panel. B A549 and H460 cells were treated with EOAI for 48 h. CASP3 activity was examined by FACS (left panel). The percentage of cells with activated caspase3 is shown in the right panel. C A549 and H460 cells were treated with EOAI for 24 h. mitochondrial membrane potential (MMP) was monitored with JC-1 dye (left panel). Cells that lost MMP appeared in the right lower quadrant and were analyzed in the right panel. The statistical significance of differences between groups in panels (A, B, and C) was assessed by the t-test of GraphPad Prism8 software. * The difference between the two groups was significant (P < 0.05). All data represent at least three independent experiments (n = 3; error bar, SD). D Detection of the expression of apoptotic, proapoptotic, and anti-apoptotic proteins after EOAI treatment for 48 h. A549 and H460 cells were treated with EOAI, and cell extracts were prepared for western blotting analysis. GAPDH and β-actin were used as the loading control. All data were representative of at least three independent experiments