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Supporting Materials

Human glioblastoma (A172) and neuroblastoma (SH-SY5Y) cell lines were obtained from American Type Culture Collection (ATCC). The culture of F11 cells was done as described (1-3). Cell culture reagents were obtained from Life Technologies (Rockville, MD) and ATCC. Humanin (HN) peptides (HN, HNG, HNA, F6A-HN, K21A-HN, and F6/K21A-HN) were synthesized by Peptides International (Louisville, KY) or Genemed Synthesis (San Francisco). Lipofectamine Plus Reagent was from Invitrogen. Ni-NTA agarose beads were from Qiagen (Hilden, Germany). M2 agarose beads were from Sigma. HN plasmids were prepared as described (1-3). pAla-scanned HN plasmids were constructed as described (3). Recombinant human insulin-like growth factor-1 (IGF-I) was provided by Amersham Pharmacia Biotech. Recombinant human IGF-binding protein-3 (IGFBP-3) and IGFBP-3 peptides were a generous gift from D. Mascarenhas (Protigen, Mountain View, CA). rhIGFBP-3 purchased from Upstate Biotechnology (Charlottesville, VA) was used in Ni-NTA and M2 pull-downs. Recombinant human ¹²⁵I-IGFBP-3 and affinity-purified anti-human IGFBP-3 antibody were purchased from DSL (Webster, TX). ¹²⁵I-HN was from Bachem, and ¹²⁵I-IGF-I was from Amersham Pharmacia Biotech. SDS/PAGE reagents, Tween, and fat-free milk were purchased from Bio-Rad. Yeast strain Saccharomyces cerevisiae HF7c and a yeast two-hybrid screening kit, including the HeLa cDNA library, were purchased from Clontech. ¹²⁵I-signals were detected and quantified by using the Storm PhosphorImager and IMAGEQUANT (Molecular Dynamics). Protein concentrations were determined by using the Bio-Rad DC Protein Assay (Bio-Rad). Rabbit polyclonal anti-HN antibody was generated by Harlan Bioproducts for Science (Madison, WI). Secondary antibodies were from Amersham Pharmacia Biotech. The SuperSignal chemiluminescent detection system was from Pierce. Hyperfilm-enhanced chemiluminescence was from Eastman Kodak. The Apo-ONE homogenous caspase-3/-7 assay and DeadEnd Colorimetric terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) system were from Promega. Amyloid b (Ab ) (1-43) peptide was from Peptide Institute (Osaka), and the Cell Counting Kit-8 was from Wako Pure Chemical (Osaka).

Yeast Two-Hybrid Screening. Briefly, the yeast strain Saccharomyces cerevisiae HF7c [MATa, ura3-52, his3-200, lys2-801, ade2-101, trp1-901, leu2-3, 112, gal4-542, gal80-538, LYS2::GAL1–HIS3, URA3::(GAL4 17 mers)3–CYC1–lacZ] was purchased from Clontech. The fusion gene IGFBP-3/binding domain (BD) was constructed by splicing cDNA encoding the human IGFBP-3 gene into the plasmid pGBT9 directly 5' to and in phase with the gene encoding the BD of the yeast transcriptional activator GAL4 (Clontech). A HeLa cDNA library with the activation domain of the GAL4 gene was purchased from Clontech and screened by cotransforming yeast with both plasmids. Yeast colonies were made competent for transformations according to the manufacturer’s instructions (Clontech Matchmaker protocol handbook). Positive cotransformants were selected by growth on histidine-deficient agar media and assayed for b -galactosidase activity. All results were reproduced in at least two additional assays. Genes encoding IGFBP-3–binding proteins identified with this method were isolated by plasmid recovery, amplified by using PCR, sequenced by using a GAL4 activation domain sequencing primer, and compared with known sequences in GenBank by using the MACVECTOR software program (Oxford).

Yeast Mating Assays. The IGFBP-3/BD was cotransformed with the candidate gene/activation domain construct into yeast by using the protocol for the two-hybrid system. The transformants were selected on leucine-, tryptophan-, and histidine-deficient SD agar plates and incubated for 3 days at 30°C. Expression of the LacZ gene by the cotransformants was determined by assaying for b -galactosidase activity according to the manufacturer’s instructions (Clontech).

Ligand Dot Blot. Western ligand blots were used to assess IGFBP-3 binding. Peptides (2 m l at a time) were carefully dot-blotted directly onto poly(vinylidene difluoride)

(PVDF) membrane (membrane presoaked in methanol and subsequently balanced in PBS) and allowed to dry completely. PVDF membrane was then rewetted with methanol, soaked in distilled H₂O, and then buffered in Tris-buffered saline (TBS)/3% igepal CA-630 with gentle shaking for 30 min at +4°C, after which the membrane was blocked with TBS/1% BSA for 3 h at +4°C. ¹²⁵I-rhIGFBP-3 (DSL) (10⁶ cpm) was incubated in TBS/1% BSA/0.1% Tween overnight at +4°C with gentle shaking. The membrane was washed with TBS/0.1% Tween four times and rinsed once with TBS. ¹²⁵I signals were detected by using the Storm PhosphorImager and IMAGEQUANT software (Molecular Dynamics).

Cross-Linking. One microgram of rhIGFBP-3 was mixed with 1 × 10⁵ cpm of ¹²⁵I-rhIGF-I (Amersham Pharmacia Biotech) and/or unlabeled rhIGF-I (1.5 m g) and/or HN or HNG peptide (1 or 5 m g) and incubated in PBS at +15°C overnight. ¹²⁵I-HN (1 × 10⁵ cpm; Bachem) was incubated with 1 m g of rhIGFBP-3 and unlabeled HN or F6A-HN (2, 1, or 0.1 m g). Disuccinimidylsuberate (DSS) at a final concentration of 0.3 mg/ml was added to the reaction and incubated for 30 min at +4°C. The reaction was stopped with 5 mM EDTA and mixed with 2´ Laemmli Sample buffer, boiled, and resolved by SDS/PAGE. The gel was dried, exposed to the STORM PhosphorImager screen, and analyzed with IMAGEQUANT software.

Pull-Down. Synthetic (His)_6x-tagged HN derivative peptides (1 nmol) were incubated with 20 m l of Ni-NTA agarose beads (Qiagen) in a total volume of 0.5 ml of buffer A [20 mM Tris· HCl (pH 8.0)/1 mM DTT] for 1 h at 4°C. The agarose beads were washed once with buffer B [20 mM Tris· HCl (pH 7.4)/1 mM DTT/150 mM NaCl]. One nanomolar rhIGFBP3 (Upstate Biotechnology) was mixed with beads immobilizing (His)_6x-tagged HN peptides in a total volume of 0.5 ml of buffer B for 2 h at 4°C on a rotating shaker, after which the beads were washed three times with buffer B. The washed beads were mixed with 20 m l of 2´ sampling buffer [100 mM Tris· HCl (pH 6.8)/200 mM DTT/4% (wt/vol) SDS/0.2% bromophenol blue/20% (vol/vol) glycerol] and boiled. The supernatant was resolved by SDS/PAGE, followed by immunoblotting analysis with polyclonal anti-IGFBP3 antibody according to standard protocols (4) and enhanced chemiluminescence detection.

Alanine Scanning. pAla-scanned HN plasmids (3) pHN or pL9R-HN (1 m g of DNA) were transfected in triplicate to F11 cells (3) (7 × 10⁴ cells per well on six-well plates) with Lipofectamine Plus. Cell lysates were precipitated with M2 antibody, and 1 or 10 nmol rhIGFBP-3 was eluted through the column according to standard protocols [72 h after the onset of transfection, transfected cells were collected and lysed with 30 m l of immunoprecipitation lysis buffer (50 mM Tris· HCl, pH 7.4/150 mM NaCl/1 mM EDTA/1% Triton X-100)] Three hundred micrograms of cell lysate and 20 m l of M2 agarose beads (after washing with TBS) were incubated with rotation in a total of 1,000 m l of immunoprecipitation lysis buffer overnight at +4°C. M2 agarose beads were washed three times with washing buffer B [20 mM Tris· HCl (pH 7.4)/150 mM NaCl/1 mM DTT]. One or ten nanomolar of rhIGFBP-3 (Upstate Biotechnology) was mixed with the beads immobilizing FLAG-fused HN peptides in a total volume of 500 m l, incubated on a rotating shaker for 8 h at +4°C, and washed three times with washing buffer B. The washed beads were mixed with 20 m l of 2´ sampling buffer [100 mM Tris· HCl, pH 6.8/200 mM DTT/4% (wt/vol) SDS/0.2% bromophenol blue/20% (vol/vol) glycerol] and boiled. The samples were resolved by SDS/PAGE, followed by immunoblotting analysis with anti-IGFBP-3 antibody and enhanced chemiluminescence detection.

Cell Culture. A172 glioblastoma and SH-SY5Y neuroblastoma cells (ATCC) were maintained and subcultured according to standard procedures. A172 were cultured in DMEM, supplemented with 10% heat-inactivated FBS, and antibiotics. SH-SY5Y were cultured in 1:1 mixture of MEM and F12 Kaighn’s modification supplemented with 10% FBS, and antibiotics. F11 cells were cultured as previously described (3).

The primary culture of mouse cortical neurons was performed in poly-L-lysine-coated 96-well plates (Sumitomo Bakelite, Akita, Japan) in the absence of serum and presence of N2 supplement, as described (3). The purity of neurons by this method was >98%.

TUNEL Staining. TUNEL staining was performed by using the DeadEnd Colorimetric TUNEL system (Promega) according to manufacturer’s instructions. Briefly, cells were plated on Lab-Tek chamber slides (Nalge Nunc, Naperville, IL) and treated with 500 nM HN or F6A-HN with or without 4 m g/ml rhIGFBP-3 in serum-free (SF) conditions for 6 h. After control or experimental treatments, the slides were washed twice with PBS and then fixed by immersing slides in 4% paraformaldehyde for 25 min at room temperature (RT). The cells were permeabilized with 0.2% Triton X-100 for 8 min at RT. The slides were incubated with TdT Reaction Mix (Promega DeadEnd Colorimetric TUNEL System) at 37°C for 60 min to allow the end-labeling reaction to occur. The reaction was stopped by immersing the slides in 2´ SSC for 15 min at RT followed by blocking of the endogenous peroxidase activity with 0.3% hydrogen peroxide for 5 min at RT. The slides were incubated with streptavidin–horseradish peroxidase for 30 min at RT and then washed three times, after which they were stained with diaminobenzidine, rinsed with deionized water, and mounted for visualization with a Nikon Labophot-2 microscope. Representative photomicrographs were taken by using a Spot digital camera and SPOT ADVANCED software (Diagnostic Instruments).

Caspase Assays. Briefly, cells were plated at equal density (10⁴ cells per ml) on 96-well plates (Costar) in medium containing 10% FBS and antibiotics. Cells were cultured overnight and changed to SF media for the experiments. rhIGFBP-3 was used at a final concentration of 1–2 m g/ml, rhIGF-I was used at a final concentration of 100–200 ng/ml, HN was used at a final concentration of at 100 nM, and phorbol 12-myristate 13-acetate (PMA) was used at a final concentration of 25 ng/ml. After the experimental and control treatments, 1:100 dilution of caspase reagent was added to cells on a 96-well plate (1:1 vol/vol reagent to sample ratio), mixed at 300 rpm for 2 min, and incubated for 30 min at RT. The fluorescence was measured by using a spectrofluorometer (Bio-Rad) with excitation at 485 and emission at 530 nm. Results are presented as relative fluorescence vs. control fluorescence at SF.

Immunoprecipitation and Immunoblotting. For the coimmunoprecipitation of HN and IGFBP-3 in vitro, A172 cells were treated with 10 m M HN overnight in SF conditions. Whole-cell extracts from HN-treated cells were prepared according to the standard protocols by lysing cells on the culture dish in 400 m l of ice-cold lysis buffer (50 mM Tris· HCl, pH 7.4/0.1% Triton X-100/1 mM EDTA/protease inhibitor mixture). For the coimmunoprecipitation of HN and IGFBP-3 in vivo, testes of 3-wk-old mice were homogenized in lysis buffer. The homogenized sample suspension was transferred by using a 27-gauge needle into a microcentrifuge tube, incubated on ice for 30 min, microcentrifuged at 10,000 ´ g for 10 min at +4°C, and the pellet was discarded. The protein concentrations were determined by using the Bio-Rad DC Protein Assay. Five hundred micrograms of total protein sample was immunoprecipitated with control goat IgG or polyclonal anti-mouse (testes) or anti-human (A172 cells) IGFBP-3. Western blotting was performed according to standard protocols. Primary and secondary antibodies were used with the following dilutions: rabbit polyclonal anti-HN antibody 1:1,000 and 1:10,000 goat anti-rabbit-IgG–horseradish peroxidase. The protein bands were visualized by using a chemiluminescent detection system (SuperSignal, Pierce).

WST-8 and Calcein Assays. Primary cortical neurons (seeded in 96-well plates: 5 × 104 cells per well, 100 m l of culture medium per well) were treated with or without 25 m M Ab (1-43) in the presence or absence of 10 Pm–10 nM HNG and/or 10 nM (0.35 m g/ml) rhIGBBP3 for 72 h. The WST-8 cell viability assay was performed by using the Cell Counting Kit-8, as described (5). The calcein assay was performed with calcein AM (Dojindo, Kumamoto, Japan), as described (3, 5). These assays were performed simultaneously. After 72 h, cells were added with the mixture of 10 m l of WST-8 solution and 1 m l of 600 m M calcein-AM. After 2-h incubation in a CO₂ incubator at 37°C, WST-8 absorbance was measured at OD 450. After changing the medium to PBS to lower the background, calcein-specific fluorescence (excitation, 485 nm; emission, 535 nm) was observed by fluorescence microscopy or measured by a spectrofluorometer (Wallac1420 ARVOsx Multi Label Counter; Perkin–Elmer).

Cloning of HN as an IGFBP-3 Partner. We used the yeast strain Hf7c (which is unable to grow in histidine-, leucine-, or tryptophan-deficient media) with a histidine-selection marker and b -galactosidase marker under control by the GAL1 promoter. The transcriptional activator GAL4 consists of two separable domains: a DNA–BD that binds to an upstream activating sequence, and a transcriptional activation domain (AD) necessary for RNA synthesis. We performed cotransformations with: (i) a pGBT9 plasmid containing the fusion gene IGFBP-3/GAL4 BD and a histidine-selection marker, plus (ii) a pGAD424 plasmid containing a HeLa cell cDNA library/GAL4 AD fusion gene and a tryptophan-selection marker. Positive cotransformants were isolated by growth on tryptophan-, leucine-, and histidine-deficient media, and positive colonies were further isolated with b -galactosidase activity and harvested to recover library plasmids. Library fragments were amplified by using PCR and sequenced.

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