Figure 5

CM transfer and co-plating experiments to evaluate bystander killing in heterogeneous system. Involvement of sFasL in mediating bystander cytotoxicity. The effector cells (THP-1 MΦ) were treated with 500 nM MMC for 24 h, and then CM was collected after 48 h as described in Materials and Methods. Target HeLa (a) and SiHa (b) cells were incubated with the CM in the presence or absence of MG132 and/or with anti-FasL antibody for 36 h. CM was supplemented with 0.2% FBS to avoid cell death owing to growth factor depletion. Cell survival was further evaluated by MTT assay. Data are mean±S.D., and are representative of three independent experiments performed in triplicates (**P<0.01 when compared with their respective controls). (ci) Apoptotic cell death in target HeLa-EGFP cells in heterogeneous system. Histograms for effector and target cell populations alone or in co-culture are shown by annexin V-PE staining using flow cytometry. 1:Untreated THP-1 MΦ; 2: Untreated HeLa-EGFP cells; 3: Untreated effector cells (THP-1 MΦ) were co-plated with target EGFP cells; 4: Effector cells (THP-1 MΦ) were treated with MMC for 24 h, washed with medium and target EGFP cells were co-plated; 5: Untreated effector cells (THP-1 MΦ) were co-plated with target EGFP cells and treated with MG132 for 24 h; 6: Effector cells (THP-1 MΦ) 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 (THP-1 MΦ) were treated with GW9662 for 2 h followed by 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 (THP-1 MΦ) 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; are shown with annexin V-PE positive counts indicated as percentages of apoptotic cells. Upper right quadrant represents proportion of apoptotic bystander target EGFP cells. Effector cells were washed with medium three times before co-plating with the target cells. (cii) 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). (d) Western blot analysis for PARP cleavage. Target HeLa cells were pretreated with 5 μM MG132 for 2 h. Thereafter, CM collected from MMC-treated effector cells (HeLa and THP-1 MΦ) was added in the presence or absence of MG132 for an additional 24 h. Whole-cell lysates were prepared to perform western blot analysis. The levels of PARP (p116) and its cleaved product (p85) were detected. β-Actin was used as a loading control