On-line Supplementary Information
Mitochondria to nucleus translocation of AIF in mice lacking Hsp70 during ischemia/reperfusion
Sangita Choudhury, Soochan Bae, Qingen Ke, Jiyoo Lee, Jacob Kim, Peter M. Kang
Supplemental Figure 1
Supplement Figure 1:Western blot analysis of cytosolic AIF and cytochrome c in WT mice after sham operation. GAPDH is the internal loading control for cytosolic fractions. There was a very modest increase in cytosolic AIF and cytochrome c after sham operation, most likely due to the effect of anesthesia on heart.
Supplemental Figure 2
Supplement Figure 2: Effect of I/R and PARP-1 inhibition on mitochondrial Bcl-2 protein levels. Western blot analysis of mitochondrial Bcl-2 in WT and Hsp70 KO mice after 45 minutes of ischemia and 9 hours of reperfusion. We also found that the level of Bcl-2, which was significantly downregulated by I/R, did not change significantly with 4-AN treatment.
Supplemental Figure 3
Supplemental Figure 3:Quantitative analysis of TUNEL staining innon-infarcted and non-ischemic areasfrom WT and Hsp70 mice after 45 minutes of ischemia and 24 hours of reperfusion.N =4. All comparisons are non-significant.
Supplemental Detailed Methods
Subcellular fractionation of heart tissues
The hearts were snap frozen in liquid nitrogen and homogenized in ice-coldbuffer A (20mM Tris·HCl (pH 7.5), 2 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethlysulfonylfluoride, 1 mM DTT, 0.3 M sucrose, 25 µg/ml leupeptin) with a Polytron electric homogenizerfor 20 sec and then with a Dounce homogenizer (60strokes). The homogenates were centrifuged at 2500 rpm for 10min at 4°C, and the supernatant was ultracentrifuged at 40,000 rpm for30 min at 4°C. The resulting supernatant was retained as the cytosolicfraction.
Immunoblots
Protein concentration was determined using the Bradford method (Bio-Rad, Hercules, CA). Samples with equal amounts of protein were separated by SDS-PAGE and transferred to an Immobilin-P transfer membrane, which was probed with specified antibodies. Visualization of the antigens was done with a chemiluminescence detection system. The western blots were quantified by the NIH Image J program.
Caspase and PARP activity assay
The hearts were snap frozen in liquid nitrogen and homogenized in caspase lysis buffer (20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, 10g/ml leupeptin, 2 g/ml aprotinin) for 15 minutes on ice. They were then mechanically disrupted and centrifuged at 15,000g for 20 minutes to obtain cell lysates. Equal amounts of protein were incubated with 50 M of caspase substrate in caspase reaction buffer mM 50 HEPES (pH 7.4), 75 mM NaCl, 0.1% CHAPS, 2 mM DTT in a 96-well microplate at 37oC for 60 minutes. Caspase-3 activities were measured using synthetic caspase substrate AcDEVD-pNa. Release of pNa was measured at 405 nm wavelength by spectrometer, and adjusted to the background. PARP activity was measured from cell extracts with an ELISA based, PARP Universal Colorimetric Assay Kit (R & D Systems, Minneapolis, MN) according to the manufacturer’s instruction.
Immunohistochemistry
Frozen 5 m-thick ventricle muscle cross sections were cut in a freezing cryostat at -20°C and placed on the same glass slide to control for processing differences (e.g., incubation time, temperature, etc.). The sections were air dried at room temperature, fixed in 4% paraformaldehyde in PBS, pH 7.4 at room temperature for 20 min, permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate at 4°C for 3 min, and incubated with fluorescein-conjugated TUNEL reaction mixture (Roche Applied Science, Indianapolis, IN) in a humidified chamber at 37°C for 1 h in the dark. Positive control experiments were performed by treating the sections with DNaseI before the TUNEL reaction mixture incubation, whereas negative control experiments were done by omitting the TdT enzyme in the TUNEL reaction mixture on the tissue sections. The sections were then blocked in 10% donkey serum in PBS at room temperature for 30 min following permeabilization with 0.5% Triton in 0.02% saponin/PBS for 10 min. After washes in PBS, sections were incubated with an anti-AIF rabbit polyclonal antibody overnight at 4oC followed by an anti-rabbit IgG rhodamin conjugated secondary antibody incubation (Sigma, St Louis, MO) for 1 h at 4oC. Negative control experiments omitted the AIF antibody from the tissue sections. The sections were then incubated with 4',6-diamidino-2-phenylindole(DAPI) staining solution (Molecular Probes, Eugene, OR) to visualize nuclei, and finally mounted with mounting medium (Vector Laboratories, Burlingame, CA). TUNEL, DAPI, and AIF staining were examined under a confocal fluorescence microscope (BioRad 1024 with Nikon E800).
Immunoprecipitation
Immunoprecipitation was performed using Catch and Release v2.0 according to the manufacturer’s instructions (Millipore, Billerica, MA). In brief, the crude protein extractions from WT and Hsp70 KO heart tissue were homogenized and incubated with anti-AIF antibody (10μl antibody per 500μg total protein) at 4°C overnight in a catch and release spin column. The bound proteins were eluted and separated on a 12% SDS–PAGE gel and subjected to western blotting using Hsp70 antibodies.
Measurement of the integrity of the outer membrane of mitochondria
Outer membrane integrity was assessed using a Cytochrome c Oxidase Assay Kit in accordance with the manufacturer’s instructions (Sigma, St. Louis, MO). Cytochrome c oxidase activity was measured in the presence and absence of the detergent, n-dodecyl b-D-maltoside, which is one of the few detergents that allow the cytochrome c oxidase dimer to be maintained in solution at low detergent concentrations. The ratio between activity with and without n-dodecyl b-D-maltoside present is a measure of the integrity of the mitochondrial outer membrane, since the membrane is a barrier for theentrance of cytochrome c into the organelle. The colorimetric assay in this kit is based on observation of the decrease in absorbance at 550 nm of ferrocytochrome c caused by its oxidation to ferricytochrome c by cytochrome c oxidase.
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