Cytosolic interaction between AIF and Scythe is required for macrophage clearance of Fas-triggered Jurkat cells.

<p>A. Immunocytochemistry analysis of Scythe and AIF in Jurkat cells un-treated and treated for 3 h with Fas mAb (250 ng/ml) in the presence or absence of the pan-caspase inhibitor, Z-VAD-FMK (20 µM). Nuclei are stained in blue (DAPI). B. Jurkat cells treated like in A were lysed and AIF was then immunoprecipitated. Western blot is 10% of the input and is derived from both control and Fas mAb treated cells. Negative control (NC) consists of glass beads incubated with normal rabbit serum. C. Jurkat cells were transfected with siRNA for AIF, Scythe and non-targeting siRNA (NC). After 48 h cells were collected, lysed in RIPA buffer and Western blotting analysis was performed to detect the levels of AIF. GAPDH was used as loading control. Quantification was made using the program Image J and the values of optical density (OD) were normalized to the levels of GAPDH. D. Jurkat cells, treated with siRNA for AIF, Scythe or NC were induced to undergo apoptosis by Fas mAb (50 ng/ml) or staurosporine (0.5 µM). After 3 h cells were co-cultivated with human monocyte-derived macrophages (HMDM) for 1 h. Examples of phagocytosis of TAMRA-labelled Jurkat cells (red) are depicted. Nuclei of macrophages and engulfed target cells are stained with DAPI (blue). E. Phagocytosis is reported as the percentages of macrophages positive for uptake of target cells. Data are presented as the mean ± S.D of six values derived from two different experiments performed in triplicate. *P<0.05; **P<0.01 (one-way ANOVA <i>Dunnett’s</i> test). Caspase-3 activation in WT versus AIF or Scythe siRNA-transfected cells determined by immunoblotting is shown in the panel below. GAPDH is used as loading control.