Figure 3.
MG132 sensitizes cervical cancer cells to FasL-mediated cell death. (ai and bi) MTT assay in MG132 and MMC-treated HeLa and SiHa cells. HeLa (ai) and SiHa (bi) cells (7 × 103/well) were seeded in 96-well plates. After 24 h, treatment of 5 μM MG132 was given for 2 h. Thereafter, 200 or 500 nM MMC was added for next 34 h, and MTT assay was performed. (aii and bii) Annexin V-FITC staining in MG132 and MMC-treated cervical cancer cells. HeLa (aii) and SiHa (bii) cells (1 × 105) were seeded in 35 mm plate. After 24 h, treatment of 5 μM MG132 was given for 2 h. Thereafter, 500 nM MMC was added for further 34 h. Next, cell were stained with annexin V-FITC, and analyzed by flow cytometry. Data are mean±S.D., and are representative of three independent experiments (*P<0.05, **P<0.01 when compared with their respective controls). (c and d) Involvement of sFasL in mediating bystander cytotoxicity. The effector cells (HeLa and SiHa) were treated with 500 nM MMC for 24 h and then CM medium was collected after 48 h as described in Materials and Methods. Target HeLa (c) and SiHa (d) cells were incubated with the respective CM in the presence or absence of MG132 and/or anti-FasL antibody or IgG control antibody for 36 h. CM was supplemented with 0.2% FBS to avoid cell death owing to growth factor depletion. Cell survival was evaluated by MTT assay. Data are mean±S.D., and are representative of three independent experiments (*P<0.05, **P<0.01, when compared with their respective controls)