Combination treatment of ionizing radiation with phytosphingosine enhances Bax translocation to mitochondria. (A) Analysis of Bax translocation by subcellular fractionation. Mitochondrial fractionation was performed with Jurkat clones treated with 10 Gy of γ-radiation alone, 5 μg/mL of phytosphingosine alone, or combination of γ-radiation (10 Gy) and phytosphingosine (5 μg/mL). After 3 hours, proteins were subjected to Western blot analysis with anti-Bax (top blot of each pair) and HSP 60 (bottom blot of each pair) antibodies. HSP 60 was used as a mitochondrial marker protein. (B) Representative confocal images for translocation of Bax to the mitochondria. Clone no. 6 was incubated with Mitotracker and stained with anti-Bax antibody and analyzed by confocal laser scanning microscopy. Mitochondrial localization of Bax was defined by yellow spots, indicating overlap of fluorescein isothiocyanate (Bax) and Mitotracker-Red. (C) siRNA targeting of the Bax attenuates combination treatment-induced cell death. The clone no. 6 transfected with Bax siRNA was treated with 10 Gy of γ-radiation alone and/or 5 μg/mL of phytosphingosine. After 3 hours, cells were stained with Hoechst 33258 and apoptotic cells were quantitated by fluorescence microscopy. Results from 3 independent experiments are shown as means ± SEM. (D) The clone no. 6 transfected with Bax siRNA was treated with 10 Gy of γ-radiation and/or 5 μg/mL of phytosphingosine. After 3 hours, mitochondrial transmembrane potential of these cells was determined by retention of DioC₃₍₆₎ added during the last 30 minutes of treatment. After removal of the medium, the amount of retained DioC₃₍₆₎ were measured by flow cytometry. (E) The clone no. 6 transfected with Bax siRNA was treated with 10 Gy of γ-radiation and/or 5 μg/mL of phytosphingosine. After 3 hours, nuclear fraction was prepared and was subjected to Western blot analysis with anti-AIF and -Ref-1 antibodies. Ref-1 was used as a nuclear marker protein.