PARP-1 activation induced by combination treatment of ionizing radiation with phytosphingosine is independent from ROS generation. (A) Radiation-resistant cells 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. After 3 hours, cell lysates were subjected to Western blot analysis with anti-PAR and β-actin antibodies. The data represent a typical experiment conducted at least 3 times with similar results. (B) 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 10 mM NAC. After 3 hours, PARP enzyme activity was measured using a commercial kit under guidance of manufacturer (see “Materials and methods”).