Figure 4.

Co-plating experiments to evaluate contact-dependent bystander killing in homogeneous system. Analysis of apoptotic cell death in target EGFP-expressing cervical cancer cells. (ai) Histograms for effector (HeLa) and target cell (HeLa-EGFP) populations alone or in co-culture are shown after annexin V-PE staining as described in Materials and Methods. 1: Untreated HeLa cells; 2: Untreated HeLa-EGFP cells; 3: Untreated effector cells were co-plated with target EGFP cells; 4: Effector cells were treated with MMC for 24 h, washed with medium and target EGFP cells were co-plated; 5: Untreated effector cells were co-plated with target EGFP cells and treated with MG132 for 24 h; 6: Effector cells were treated with MMC for 24 h, washed with medium, and target EGFP cells were co-plated and then treated with MG132 for 24 h; 7: Effector cells were treated with GW9662 for 2 h followed by the addition of MMC, for 24 h, and then washed, co-plated with target EGFP cells along with treatment of MG132 and GW9662; 8: Effector cells were treated with MMC for 24 h, washed and co-plated with target EGFP cells, and further incubated in the presence of MG132 and anti-TRAIL antibodies; are shown with annexin V-PE positive counts and are indicated as percentages of apoptotic cells. Upper right quadrant represents proportion of apoptotic target EGFP cells. (aii) The bar graph shows annexin V-PE positive cells in the same experiment. Data are mean±S.D., and are representative of three independent experiments (**P<0.01, when compared with their respective controls). (bi) Similar experiment was performed in SiHa cells (effector SiHa and target SiHa-EGFP). (bii) The bar graph shows annexin V-PE positive cells in the same experiment. Data are mean±S.D., and are representative of three independent experiments (**P<0.01, when compared with their respective controls)