Figure 7

Expression of death receptors increases after MMC treatment in hepatoma cells. HepG2 and Hep3B cells were treated with MMC for 24 h. Cells were washed twice with medium, fresh medium without MMC was added and cells were incubated again for 24 h at 37°C. After post-treatment growth in MMC free medium whole cell lysate was prepared and western blot was performed. (A) Western blot analysis: lane (1) control HepG2 cells, lane (2) HepG2 treated for 24 h with MMC and lane (3) HepG2 treated for 24 h with MMC followed by post treatment growth in drug free medium for 24 h. β-actin was detected as a loading control. Where ever required, blots were stripped by incubating the membranes at 50°C for 30 min in stripping buffer (62.5 mM Tris-Cl pH 6.7, 100 mM mercaptoethanol, 2% SDS) with intermittent shaking. Membranes were washed thoroughly with TBS and reprobed with required antibodies. Otherwise gels run in duplicates were probed for the desired proteins by western blotting. (B) Immunofluorescence staining of HepG2 cells. Cells were treated with MMC for 24 h, washed twice with medium and fresh medium without MMC was added and cultured for 24 h. Cells were fixed with 4% paraformaldehyde, permeabilized with 1% Triton X- 100 and blocked with 5% FBS. Cells were then incubated with anti-FasL, anti-Fas, anti-TRAIL and anti-DR4 primary antibodies for 2 h and subsequently stained with FITC conjugated secondary antibody for 1 h.