Figure 4

AA005 promotes translocation of AIF to cell nuclei. (A) Flow cytometry analysis of AA005- or MNNG-induced cell death in the presence of PARP inhibitor Olaparib (AZD2281; 50 μM). Panel numbers show percentages of Annexin-V⁺ or PI⁺ cells. Lower: dead cells. Each column shows mean ± S.D. of triplicates in an independent experiment. **P < 0.01, versus AA005- or MNNG-treated group. (B) Analysis of AIF translocation by subcellular fractionation. SW620 cells 48 hours after AA005 treatment, 8 hours after MNNG treatment or control cells were subjected to subcellular fractionation; immunoblotting was performed with nuclear and mitochondrial fractions. Lamin B and COX IV were used as nuclear and mitochondrial marker proteins, respectively. (C) NB4 cells were treated with or without 1 μM AA005 for indicated times and subjected to subcellular fractionation; immunoblotting was performed with nuclear and mitochondrial fractions. Lamin B and COX IV were used as nuclear and mitochondrial marker proteins. (D) SW620 cells grown on chamber slides were treated with or without 1 μM AA005 or 500 μM MNNG. Subcellular localization of AIF protein was examined by Olympus BX-51 fluorescence microscope analysis. DAPI: DAPI nuclear counterstaining. > 100 cells were inspected per experimen; cells with typical morphology are shown. Scale bars, 20 μm. The experiments in B–D were repeated at least three times with similar results.