Figure 3

AIF translocation to the nucleus triggers ESA-mediated apoptosis together with ERK. (a) AIF was localized in the nucleus in PC12 cells 5 h after ESA stimulation as shown in 3D images. AIF, nuclei and DNA cleavage by TUNEL were labeled in green, blue and red, respectively. Informative illustration shows that TUNEL staining overlapped with AIF. (b) AIF translocation was observed in FBD-102b cells and rat cortical neurons. Images were obtained from cells treated with ESA for 5 h (FBD-102b) or 48 h (rat neurons). (c) Subcellular fractionation experiments show the release of AIF, COX Vα and Mn-SOD into the cytoplasm. (d) Deletion mutant ΔN102-AIF, which lacks 102 amino acids at the N-terminus, did not induce cell death by itself, although it was expressed in the nucleus. (e) Cyp-A is not necessary for ESA-mediated apoptosis. ΔN102-AIF-GFP did not initiate cell death, despite colocalization of AIF and endogenous Cyp-A in the nucleus. siRNA for Cyp-A was not able to suppress ESA-mediated apoptosis in PC12 cells. Values are the means±S.D. (n=3). NS represents not significant. (f) Lep-B, which is forced to induce the nuclear localization of ERK1/2, exacerbated cell viability only in ΔN102-AIF-transfected cells. Partial colocalization of both AIF and ERK1/2 was seen ESA-treated cells. The presence of AIF and ERK1/2 in the nucleus appears to be necessary for ESA-mediated apoptosis. Experiments were performed in two transfection conditions, lipofection and electroporation. Cell viability data were obtained from two independent experiments in triplicates.