Enhancement of PARP-1 activation by combination treatment of ionizing radiation with phytosphingosine. (A) Clone no. 6 was 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 30 μM DPQ or PARP-1 siRNA. After 3 hours, PARP enzyme activity was measured using a commercial kit under guidance of manufacturer (see “Materials and methods”). (B) Radiation-resistant cells were treated with 10 Gy of γ-radiation and/or 5 μg/mL of phytosphingosine in the presence or absence of 30 μM DPQ or PARP-1 siRNA. After 3 hours, cell lysates were subjected to Western blot analysis with anti-PARP-1 and β-actin antibodies. The data represent a typical experiment conducted at least 3 times with similar results. (C) Radiation-resistant cells were treated with 10 Gy of γ-radiation and 5 μg/mL of phytosphingosine in the presence or absence of 30 μM DPQ. 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 amounts of retained DioC₃₍₆₎ were measured by flow cytometry. (D) Radiation-resistant cells were treated with 10 Gy of γ-radiation and/or 5 μg/mL of phytosphingosine in the presence or absence of 30 μM DPQ. 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. (E) Clone no. 6 was treated with 10 Gy of γ-radiation and/or 5 μg/mL of phytosphingosine in the presence or absence of 30 μM DPQ and 10 mM NAC. After 3 hours, mitochondrial or nuclear fractions were prepared. Mitochondrial protein fraction was subjected to Western blot analysis with anti-Bax and -HSP 60 antibodies, and nuclear protein fraction was subjected to Western blot analysis with anti-AIF and -Ref-1 antibodies. HSP 60 and Ref-1 were used as mitochondria and nuclear marker proteins, respectively.