American Society of Hematology

Figure 2.

$Figure 2. Combination treatment of phytosphingosine with ionizing radiation induces AIF translocation to nucleus. (A) Analysis of AIF translocation by subcellular fractionation. Nuclear fraction was obtained from the Jurkat clones treated with 10 Gy of γ-radiation alone, 5 μg/mL of phytosphingosine alone, or combination of γ-radiation (10 Gy) and phytosphingosine (5 μg/mL) and was subjected to Western blot analysis with anti-AIF (top blot of each pair) and -Ref-1 (bottom blot of each pair) antibodies. Ref-1 was used as a nuclear marker protein. (B) Representative confocal images for translocation of AIF to the nucleus and nuclear condensation after combination treatment in clone no. 6 in the absence or presence of 30 μM z-VAD-fmk. The nuclear translocation of AIF is demonstrated by the overlap of AIF (green) and nuclear staining (red), as noted by yellow color. (C) siRNA targeting of AIF attenuates combination treatment-induced cell death. The clone no. 6 transfected with AIF siRNA was treated with 10 Gy of γ-radiation and/or 5 μg/mL of phytosphingosine. After 3 hours, cells were stained with Hoechst 33258, and apoptotic cells were quantitated by fluorescence microscopy. Results from 3 independent experiments are shown as means ± SEM.$

Combination treatment of phytosphingosine with ionizing radiation induces AIF translocation to nucleus. (A) Analysis of AIF translocation by subcellular fractionation. Nuclear fraction was obtained from the 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) and was subjected to Western blot analysis with anti-AIF (top blot of each pair) and -Ref-1 (bottom blot of each pair) antibodies. Ref-1 was used as a nuclear marker protein. (B) Representative confocal images for translocation of AIF to the nucleus and nuclear condensation after combination treatment in clone no. 6 in the absence or presence of 30 μM z-VAD-fmk. The nuclear translocation of AIF is demonstrated by the overlap of AIF (green) and nuclear staining (red), as noted by yellow color. (C) siRNA targeting of AIF attenuates combination treatment-induced cell death. The clone no. 6 transfected with AIF siRNA was treated with 10 Gy of γ-radiation 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.

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