Supplementary Fig. 1. Redistribution of apoptosis inducing factor (AIF) from mitochondria in cortical neurons treated with hydrogen peroxide (H2O2) for 4 h. Localization was determined by immunostaining for AIF and counterstaining with DAPI, followed by confocal microscopy. Upper row shows untreated (Untr) neurons(white arrows) indicating examples of cells with AIF localized in mitochondriaand aclearly visible nuclear void. Lower row shows H2O2-treated neurons(yellow arrows) indicating examples of cells with AIF redistributed throughout, including the region of the nucleus. Bars represent 10 µm.
Supplementary Fig. 2. TUNEL labeling of nuclei in cortical neurons treated with staurosporine (STS) or hydrogen peroxide (H2O2) for 24 h. The treated cells had been previously transfected with non-silencing siRNA (nRNA), or siRNA to suppress Endo G (siEndoG) expression. (A) Images of neurons stained with TUNEL to monitor cells with DNA fragmentation. DAPI was used as a nuclear counterstain to determine the total number of cells present in each field. For nRNA-transfected neurons, TUNEL-positive nuclei that are condensed under DAPI staining are indicated (white arrows). For siEndo G-transfected neurons, TUNEL-positive nuclei that are condensed under DAPI staining are indicated (yellow arrows), and TUNEL-negative nuclei that have normal morphology are indicated (red arrows).Bars represent 10 µm. (B) Quantitative analysis of TUNEL labeling in transfected cells after treatment. Black bars, nRNA; white bars,siEndoG. Results shown here report triplicate analyses within a single experiment. Due to the variation in Endo G suppression achieved between experiments (see main text), similar data obtained from three replicate experiments were not combined for quantification here.Asterisks indicate time point where there was significant difference between nRNA and siEndoG populations (P<0.001).
Supplementary Fig. 3. Cell death of cortical neurons,under conditions of AIF suppression using siRNA (siAIF), in culture medium without anti-oxidants. (A) Western immunoblot of AIF in cell lysates, with β-actin as loading control. Lanes indicate untransfected control cells (Ctrl), and cells transfected with either non-silencing RNA (nRNA) or siAIF. Representative gel shown here indicates 60% suppression of AIF by siAIF. (B) PI uptake in untreated neurons transfected with siAIF, cultured in Neurobasal medium (NBM; left panel) and minimum essential medium (MEM; right panel). DIC images are shown to visualize the total number of cells per each field. Bars represent 10 µm. (C) Quantitative analysis of PI uptake in transfected cells cultured in NBM and MEM. Black bars, NBM; white bars, MEM. Results shown here report triplicate analyses within a single experiment. Due to the variation in AIF suppression achieved between experiments (see main text), similar data obtained from three replicate experiments were not combined for quantification here.Asterisk indicates condition where there was significant difference between nRNA and siEndoG populations (P<0.0001).
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