streptomycin. Cells were incubated in a 5% CO2 humidified environment at 37uC. Cells were plated in fresh medium in optical bottom 96-well plates (Nunc) at a density of 2,500 cells/well for cytotoxicity assays. After 24 hours, media was removed and 100 mL/well fresh medium containing protein was added. Two independent experiments with triplicate wells were used for each condition. Cells incubated in media containing buffer (B) only (50 mM Tris-HCl pH 8.5, 150 mM NaCl for soluble aggregates and dimer; 0.005 mM HCl, pH 5.3 for protofibrils and fibrils) were used as a control. Concentrations of myostatin samples used in the assay were comparable to those from previous protein fibril studies [41,42,65]. WST-1 Colorimetric Assay for Cytotoxicity The WST-1 reagent (Roche) is a tetrazolium-based salt that can be metabolized by viable cells only to produce a water-soluble formazan product measurable in a colorimetric assay. After a 24- hour incubation with protein, 10 mL WST-1 was added to each well and the plate was incubated for a further 3 hours. Absorbance at 450 nm was measured in a PowerWave XS 96-well plate reader (BioTek Instruments, Inc) with 630 nm readings taken as reference. Phenol red-free media was not used as the indicator does not interfere with the WST-1 assay; as phenol red absorbs slightly at 450 nm however, wells containing media only were used as an additional control [82]. Supporting Information Figure S1 In silico predictions of propensity for b-sheet aggregation (A and B) and regions responsible for amyloid formation (C) by MstnPP. (A) Tango; (B) PASTA; and (C) Waltz. The MstnPP amino acid sequence from residues 21–375 were used for the calculations at a theoretical pH of 7. Default settings were used for all algorithms. References 1. Joulia-Ekaza D, Cabello G (2006) Myostatin regulation of muscle development: molecular basis, natural mutations, physiopathological aspects. 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(2003) Activation of latent myostatin by the BMP-1/tolloid family of metalloprotei- nases. Proc Natl Acad Sci 100: 15842–15846. Myostatin Amyloid Formation Found DOC) at: doi:10.1371/journal.pone.0009170.s001 (0.24 MB Figure S2 Western blot and SDS-PAGE of MstnPP purification procedure. (A) 12% reducing (+ bME) vs non-reducing (2 bME) SDS-PAGE and (B) subsequent Western blot of purification procedure. Lanes are as follows: H1, heparin peak 1; H2, heparin peak 2; PD, gel filtration purified dimer; PA, gel filtration purified aggregates. Major bands are indicated: 1. soluble aggregates; 2. dimer; 3. monomer. b-ME concentration in + b-ME lanes is 2 M. Found at: doi:10.1371/journal.pone.0009170.s002 (1.42 MB DOC) Figure S3 CD thermal denaturation of MstnPP soluble aggregates at pH 5.3 shows a gradual transition from an a-helical dominated spectrum at 10uC to one rich in b-sheet at 65uC. Significant loss of the a-helical minimum begins at 40uC. 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Proc Natl Acad Sci 95: 14938–14943. PLoS ONE | www.plosone.org 11 February 2010 | Volume 5 | Issue 2 | e9170