MMC induced apoptotic factors. (A) HepG2 and Hep3B cells treated with MMC were harvested and cell lysates were prepared. The expression of apoptosis related proteins were dectected by western blot analysis. The protein levels of PARP p116 proform and its p85 cleavage product, pro caspase-3 p32 and its cleavage product p11, and BID p21 proform were detected. Lane (1) untreated cells, (2) cells treated with MMC for 24 h and (3) cells treated with MMC for 24 h followed by post treatment growth in MMC free medium for 24 h. β-actin was detected as a as loading control. Where ever possible blots were stripped and reprobed with required antibodies. Otherwise gels run in duplicates were probed for the desired proteins by western blotting. (B) TUNEL assay for effector and target cells. For effector cells, HepG2 and Hep3B cells were treated with MMC (150 nM) for 24 h. Then the cells were washed with medium and cultured for additional 72 h before performing TUNEL assay. Similarly, for target cells, medium form untreated as well as from treated HepG2 or Hep3B cells were used to culture target HepG2 and Hep3B cells respectively for 48 h before performing TUNEL assay. The medium was supplemented with 0.2% FBS to avoid growth factor depletion.