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SUPPLEMENTAL CONTENT 1
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Supplemental Methods
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TUNEL stainings
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For TUNEL (terminal deoxynucleotidyl transferase (dUTP) nick end labeling) assay, snap-
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frozen tissue samples were embedded in OCT Tissue Tek compound (Sakura Finetek Europe
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B.V, Zoeterwoude, Netherlands), cryosectioned at 4 μm, and fixed in 4% formaldehyde (Pol-
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ysciences Inc., Warrington, PA, USA) in PBS for 10-15 minutes at RT, followed by permeabili-
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zation with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in 0.1% sodium citrate in
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+4C for 2 min. TUNEL reaction was performed using a Fluorescein In Situ Cell Death Detection
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Kit (Roche Diagnostics GmbH, Mannheim, Germany) according to manufacturer’s instructions.
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The sections were mounted in Vectashield with DAPI (Vector Laboratories Inc., Burlingame,
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CA, USA). Stainings were microphotographed with Axioplan2 fluorescence microscope and
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camera (Carl Zeiss Vision GmbH, Aalen, Germany) with appropriate filters. Pseudo-colored
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images were created with Axiovision 4.8 (Carl Zeiss) and Image J (NIH software). TUNEL and
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alpha-smooth muscle actin (αSMA) double stainings were performed by staining the samples
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with monoclonal anti-αSMA (1A4) antibody and Alexa 555 conjugated secondary antibody as
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described below after the TUNEL staining.
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Immunofluorescence
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Cryosections (4 μm) were fixed in 4% formaldehyde (Polysciences Inc., Warrington, PA, USA)
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in PBS for 10-15 minutes at RT, incubated in 0.1% Triton X-100 (Sigma-Aldrich, St. Louis,
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MO, USA) in PBS for 10 minutes and blocked with 3% normal goat serum (NGS) (Vector La-
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boratories Inc., Burlingame, CA, USA), 3% bovin serum albumin (BSA) (Sigma-Aldrich) in
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PBS for 30 min or 5% NGS in PBS for 60min. The anti-cleaved caspase-3 or HO-1 antibodies
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were diluted in 1.5% NGS/PBS and incubated overnight at +4°C. The primary antibody was de-
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tected with fluorochrome-conjugated Alexa Fluor 488 (green) or 555 (red) secondary antibody
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(Molecular Probes Inc., Eugene, OR, USA) diluted in 1:200 in 1.5% NGS/PBS.
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In double immunofluorescence stainings the samples first underwent staining as described above,
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followed by incubation with a monoclonal αSMA antibody (clone 1A4, Sigma-Aldrich, St Louis,
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MO, USA) diluted in 1.5% NGS in PBS. The anti-αSMA antibody was detected with Alexa 555
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conjugated anti-mouse secondary antibody, followed by background staining and mounting with
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DAPI containing Vectashield medium (Vector Laboratories Inc, Burlingame, CA). In double
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immunofluorescence stainings with the polyclonal rabbit anti-HO1 antibody, a directly Cy3-
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conjugated monoclonal anti-αSMA antibody was used (clone 1A4).
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Western Blotting (WB)
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Aneurysm samples were pulverized in liquid nitrogen using mortar and pestle, homogenized in
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hot 1% sodium dodecyl sulphate (SDS) in phosphate-buffered saline (PBS) buffer, and sonicat-
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ed. Then samples were kept at 100°C for ten minutes and immediately frozen on dry ice. The
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mixture was centrifuged at 14 000 g for 15 minutes at +4°C and the supernatant was separated.
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The protein concentration was determined using DC protein assay (Bio-Rad Laboratories, Hercu-
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les, Calif) according to the manufacturer’s instruction. For gel electrophoresis, the samples were
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mixed (5:1) with sample buffer (0.3 mol/L Tris, pH 6.8, 10% SDS, 25% β-mercaptoethanol, 50%
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glycerol, and bromophenol blue). Proteins were separated in 12% SDS polyacrylamide gel with
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Tris-glycine-SDS running buffer. Then proteins were transferred to polyvinylidene difluoride
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membrane (Bio-Rad Laboratories) with Tris-glycine transfer buffer and wet blotter (Bio-Rad
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Laboratories). The membrane was blocked with 5% nonfat dry milk in 0.1% Tween-20 (Sigma-
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Aldrich Chemie GmbH, Buchs, Switzerland) in Tris-buffered saline (TBST) for 1 hour at room
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temperature (RT). After blocking, the blots were incubated with primary antibodies against HO-
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1, caspase-8, caspase-9, caspase-3 (Supplemental Table S1) diluted in 5% BSA (Sigma-Aldrich)
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in TBST or 5% nonfat dry milk in TBST, followed by 1-hour incubation with peroxidase conju-
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gated goat anti-mouse/anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratoried
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Inc., West Grove, PA, USA) diluted 1:50 000 in milk/TBST. Antibody-antigen complexes were
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visualized by chemiluminescence using ECL Plus Western Blotting Detection Reagents (Amer-
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sham Biosciences/GE Healthcare, Piscataway, NJ) according to manufacturer’s instructions and
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Typhoon scanner (Amersham).
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Immunohistochemistry
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For immunohistochemical stainings, 4 μm tissue sections were fixed, blocked, and incubated
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with primary antibody (rabbit polyclonal against HO-1 or mouse monoclonal anti-CD45, clone
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2B11+PD7/26) as described above for immunofluorescence. The endogenous peroxide was
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blocked with 3% hydrogen peroxide in PBS. The sections were then incubated with biotinylated
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secondary antibodies (Vector) diluted in 1:200 in 1.5 % NGS or normal horse serum in PBS for
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30 min at RT followed by incubation with horseradish peroxidase-conjugated avidin-biotin com-
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plexes and visualized with diaminobenzidine (Vector). The background was stained with May-
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er’s hematoxylin. The stainings were microphotographed with Axioplan 2 light microscope and
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camera (Carl Zeiss). Image J software (NIH, USA) was used to reconstruct the aneurysm wall
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from the serial microphotographs.