Figure 2. Intrinsic pathway in IRF-1 induced apoptosis.

(a) Ad-IRF-1 induces mitochondrial release of cytochrome c. After infection (MOI 50) cells were harvested at 12, 24, and 30 h and fractionated. Cytosolic and mitochondrial fractions were then analysed by western blot for cytochrome c. VDAC is used as confirmation of successful and equivalent extraction of mitochondrial fraction. (b) AGS cells were transfected with nothing as control, non-targeting siRNA or caspase 8 siRNA (25nM) for 48 h, then the cells were infected with NI, Ad-Null or Ad-IRF-1 (MOI 50) for 30 h. Apoptosis was detected by FACS analyses as described in Materials and Methods. (c) AGS cells were subjected to the indicated treatment as in (b) for 48 h, and caspase-8 and caspase-3 were analysed by immunoblotting. Control = C, Non-targeting siRNA = N, and Caspase 8 siRNA = “8”. (d) The levels of caspase-9, Bid and cytochrome c were detected by immunoblotting with the indicated treatments in AGS cells. (e) AGS cells were transfected with nothing as control, non-targeting siRNA or Bid siRNA (25nM) for 48 h, then the cells were infected with NI, Ad-Null and Ad-IRF-1 (MOI = 50) for 30 h. Apoptosis was detected by FACS analyses as described in Materials and Methods. (f) AGS cells were subjected to the indicated treatment as in (e), and Bid, IRF-1, caspase 3 and caspase 9 were analysed by Western blotting. Control = C, Non-targeting siRNA = N, and Bid siRNA = B. (g) The levels of cytochrome c were assessed by Western blot in cytosolic and mitochondrial fractions.