Figure 8

Secreted form of FasL involved in mediating bystander cytotoxicity and apoptosis in HepG2 cells. (A) Effector HepG2 cells were pretreated with EDTA (2 μM) for 1 h and MMC was added in the same medium containing EDTA. Subsequently, this medium was used to culture target HepG2-EGFP cells and fluorescent intensity was measured after 48 h. (B) Fluorescent photomicrograph: (a) untreated HepG2 effector cells co-plated with target HepG2-EGFP cells and (b) effector HepG2 cells pretreated with 2 μM EDTA followed by MMC treatment for 24 h. (C) Sandwich ELISA to detect FasL in CM from MMC treated and cells pretreated with EDTA followed by MMC treatment for 24 h. Data presented are representative of three independent experiments performed in triplicates and expressed as mean ± s.d. ** differs significantly at p < 0.005. (D) Effector HepG2 cells treated with MMC for 24 h as well as from effector HepG2 cells pretreated with EDTA for 1 h followed by MMC treatment for 24 h. Cells were then washed twice with medium, target HepG2-EGFP cells were co-plated and the co-culture were grown for 72 h, and whole cell lysates were made from the co-culture to perform western blot analysis. The levels of PARP p116 proform and its cleavage product p85 were detected. β-actin was detected as a loading control. Lane (1) untreated effector HepG2 cells. (2) MMC treated effector cell and harvested after 24 h. (3) MMC treated effector cells and harvested after 48 h. (4) EDTA (2 μM) pre-treatment followed by MMC treatment of effector cells and harvested after 24 h. (5) EDTA (2 μM) pre-treatment followed by MMC treatment of effector cells and harvested after 48 h.