Figure 1

MMC induces expression of death ligands in cervical cancer cells. (a) Bystander killing in CM transfer experiment. The effector cells (HeLa and SiHa) were treated with indicated concentrations of MMC for 24 h, and then CM medium was collected after 48 h as described in Materials and Methods. Target HeLa (i) and SiHa (ii) cells were incubated with the respective CM for 24 h. Cell survival was evaluated by MTT assay. (b) Semi-quantitative RT-PCR for FasL mRNA. HeLa and SiHa cells were treated with indicated concentrations of MMC for 24 h, and were processed for RT-PCR. β-Actin was used as a loading control. Data are mean±S.D., and are representative of three independent experiments. (c) Western blot analysis of FasL. HeLa and SiHa cells were treated with indicated concentrations of MMC for 24 h, and cell lysates were subjected to SDS-PAGE and probed for protein levels of FasL. (d) Flow cytometric analysis of FasL expression. HeLa (i) and SiHa (ii) cells were treated with MMC as described above. Untreated or MMC-treated cells were probed with FasL primary antibody or IgG control (1 : 100), and further with PE-conjugated secondary antibody (1 : 200). Cells were then washed with PBS, and FasL expression was analyzed by flow cytometry. (e) Sandwich ELISA for quantification of sFasL from MMC-treated HeLa (i) and SiHa (ii) cells at the indicated time points. Data are mean±S.D., and are representative of three independent experiments (**P<0.01 when compared with their respective controls)