Fig. 4. The pro-apoptotic activity of ATAP involves targeting to mitochondria. (A) Schematic representation of the GFP-ATAP constructs in which point mutations were introduced into the flanking regions of the ATAP. (B) Cell survival was measured by PI exclusion in the HEK293 cells transfected with 1 µg GFP-ATAP mutant constructs 24 hours after transfection. (C) Subcellular localization of ATAP mutants fused with GFP. HeLa cells were transfected with 0.5 µg of the indicated plasmids. Cell culture was performed in the presence of 50 µM OPH to prevent rapid cell death. 18 hours after transfection, cells were incubated in medium containing 50 nM MitoTracker for 2 hours and fixed using 4% paraformaldehyde. Localization of GFP fusion proteins was observed using confocal microscopy. Bar, 10 µm. (D) Cellular effects of GFP-ATAP mutants on apoptosis. HEK293 cells were transiently transfected with the indicated plasmids expressing ATAP mutants fused with GFP. 18 hours after transfection, cells were harvested, fixed, and stained with PI. The DNA content of GFP-positive cells was then analyzed by flow cytometry (upper panel). DNA fragmentation was also analyzed by electrophoresis on 2% agarose gels (lower panel). SM, 100 bp ladder size marker. (E) Time-course effect of GFP-ATAP and GFP-ATAP mutants on HEK293 cells. Cell survival was measured by PI exclusion in the HEK293 cells transfected with 1 µg GFP-ATAP mutant constructs.