Figure 4.

Apoptosis-induced processing and mitochondrial release of AIF. (A) Schematic representation of the AIF constructs and their intracellular location. (B) Subcellular distribution of various AIF constructs in response to actinomycin D treatment. HeLa cells expressing the indicated constructs (48 h after transfection) were treated with or without actinomycin D for 12 h in the presence of zVAD-fmk. The cells were examined by fluorescence confocal microscopy for GFP. The cells with a cytoplasmic GFP pattern were counted. Each histogram indicates mean±s.d. in three fields of at least 100 cells within a representative experiment. Magnification, × 630; bar=20 μm. (C) HeLa cells expressing the indicated constructs were treated with or without actinomycin D in the absence or presence of zVAD-fmk. Total cell lysates were subjected to SDS–PAGE followed by immunoblot analysis using anti-FLAG antibody. The lysate from N120ΔN101-GFP-FLAG-expressing cells was used as a size reference (lane 3). (D) HeLa cells expressing the indicated constructs were treated with or without actinomycin D in the presence of zVAD-fmk. The cells were fractionated into membrane and cytosolic fractions, and each fraction was analyzed by immunoblotting using antibodies against FLAG (for AIF constructs) or against the indicated proteins. Open and closed arrowheads indicate the GFP fusion proteins corresponding to unprocessed and processed forms, respectively. Asterisks represent uncharacterized minor bands detected in the absence of zVAD-fmk. Note that processed forms of N120 and N110 were detectable in mock-treated cells (closed arrowhead in lanes 2 and 6). Apoptosis might be brought about in the cells to a slight extent by mock treatment with Lipofectamine to induce this processing.