Landshamer Et Al.: Bid-Induced Release of AIF from Mitochondria

Landshamer et al.: Bid-induced release of AIF from Mitochondria

Supplement Figures

Supplement Fig. 1. Therapeutic window of Bid inhibition

Quantification of cell death by MTT assay 18 h after onset of glutamate-treatment (3 mM) HT-22 neurons. BI-6C9 was applied together with glutamate (0 h) or up to 12 h afterwards. The graph shows mean values and S.D. of n=8 independent experiments per group; ***p<0.001 compared to glutamate treatment.

Supplement Fig. 2. BI-6C9 prevents tBid-induced cell death.

HT-22 neurons were pretreated with BI-6C9 (10 µM) before transfection with a tBid expressing plasmid (p-tBid). Twenty four hours later cell viability was determined with the MTT assay. ***p<0.001 compared to controls and BI-6C9-treated cells (ANOVA, Scheffé’s).

Supplement Fig. 3. Caspase-8 activation is not involved in glutamate neurotoxicity.

A, Western blot analysis of protein extracts of HT-22 cells exposed to glutamate (2-4 mM) for 17 h did not show Caspase-8 fragmentation. B, The caspase-8-activity assay did not reveal altereations of caspase-8 activity after BI-6c9 (10 µM) or glutamate-treatment (4 mM, 17 h). C, MTT-assay revealed that IETD-FMK (50 µM) could not protect HT-22 neurons from glutamate-induced cell death.

Supplement Fig. 4. Immunocytochemistry confirms that glutamate-induced cell death involves endogenous AIF translocation to the nucleus.

Confocal Fluorescence photomicrographs of HT-22 neurons show co-localization of AIF (immunostained, green) and the DAPI-signal in glutamate-damaged (4 mM, 17 h) cells. Bid inhibitor BI-6C9 (10 µM) prevented the AIF translocation to the nucleus.

Supplement Fig. 5. Neither Bid nor AIF are required for staurosporine-induced apoptosis in HT-22 cells.

A, FITC-labeled Annexin-V binding in HT-22 cells 18 h after exposure to staurosporine (300 nM) was quantified by FACS analysis. The Bid inhibitor BI-6C9 (10 µM, 1 h prior to damage) did not reduce apoptosis induced by STS. B, Cell viability of HT-22 was determined by MTT assay 18 h after treatment with STS (300 nM). Pretreatment of HT-22 cells with 10 µM BI-6C9 1 h prior to onset of STS treatment did not attenuate cell death. C, Cells were transfected with mutant siRNA or AIF-siRNA (20 nM, 48 h) before STS (300 nM, 18 h) exposure. Cell viability in STS-exposed cultures was significantly decreased by STS and was not influenced by AIF-siRNA. D, Staurosporine does not induce nuclear translocation of AIF in HT-22 cells. HT-22 cells expressing AIF-GFP fusion protein (green) were treated with STS for 18 h, before staining with DAPI (blue) and analysis by confocal fluorescent microscopy. The Bid inhibitor BI-6c9 does not prevent DNA condensation and nuclear fragmentation of HT-22 cells, and AIF does not translocate to the nucleus of the apoptotic cells.

Supplement Fig. 6: The Bid inhibitor BI-11A7, a BI-6C9 derivative, prevents nuclear translocation of AIF and protects against amyloid-beta induced cell death.

Neurons were treated with the BI-6c9 derivate and Bid inhibitor BI-11A7 in serum-free Earles-balanced salt solution (EBSS) and then exposed to amyloid-beta peptide. After 18 h apoptotic cells were quantified and AIF translocation was analysed in nuclear protein extracts by Western blots. **p<0.01 compared to amyloid-beta-treated vehicle controls.

Supplement Movie. Glutamate treatment of HT-22 cells induces translocation of AIF from mitochondria to the nucleus. The movie was composed from fluorescence photomicrographs taken every 5 minutes from 7 h to 14 h after onset of glutamate-treatment (3 mM) in HT-22 neurons expressing an AIF-GFP fusion protein. The movie shows accumulation of AIF around the nucleus in a damaged cell within 10 h after glutamate exposure. After accumulation around the nucleus, AIF is released from mitochondria and translocates to the nucleus within 10-15 minutes. This translocation of AIF is followed by chromatin condensation and fragmentation of the cell. Co-localization of AIF-GFP- (green) and DAPI-signals (dark blue) reveal a nuclear localization of AIF (merged color: light blue).