Myostatin Amyloid Formation Figure 4. Characterisation of MstnPP amyloid fibrils by ThT binding and TEM. A. ThT binding after incubation at 60uC and pH 5.3 compared to insulin and MstnPP soluble aggregates. Each result represents the average of four independent experiments and error bars represent SEM. ** P,0.005 and * P,0.01 by paired Student’s t-test. Blank is 0.005 mM HCl, pH 5.3 and ThT. (B–F) TEM showing different stages in the formation of prefibrillar structures and fibrils by human MstnPP. B. Overnight incubation at 60uC in pH 5.3 produces ‘beaded’ prefibrillar structures; C. (i) By three days large, dense three-dimensional arrays show up as areas of high electron density, (ii–v) zoomed in examples of lateral association and longitudinal fusion (ii and iii) and pore-like structures (iv and v) from (i); D. Characteristic amyloid fibrils after one week; E–F. Formation of higher order structures with the twisting of two (E) or more (F) fibrils around each other. doi:10.1371/journal.pone.0009170.g004 PLoS ONE | www.plosone.org 6 February 2010 | Volume 5 | Issue 2 | e9170