Enhancement of ROS production by combination treatment of ionizing radiation with phytosphingosine. Radiation-resistant Jurkat clones were 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) in the presence or absence of 10 mM NAC. (A) After 3 hours, cells were incubated with 10 μM of H2DCF-DA for 30 minutes and analyzed by flow cytometry as described in “Materials and methods.” (B) Mitochondrial transmembrane potential 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. (C) 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) Clone no. 6 was treated with 10 Gy of γ-radiation and/or 5 μg/mLof phytosphingosine in the presence or absence of 10 mM NAC. After 3 hours, nuclear fraction was prepared and subjected to Western blot analysis using anti-AIF or -Ref-1 antibody. Ref-1 was used as a nuclear marker protein. (E) Clone no. 6 was treated with 10 Gy of γ-radiation and/or 5 μg/mL of phytosphingosine in presence or absence of 10 mM NAC. After 3 hours, mitochondrial fraction was prepared and was subjected to Western blot analysis with anti-Bax and -HSP 60 antibodies. HSP 60 was used as a mitochondrial marker protein.