Fig 1.
Fibrillation kinetics of Aβ(1–40) aggregation in bicine solution.
ThT fluorescence assay was used to examine the propensity of fibril formation affected by bicine concentration (a) and incubation time (b). (a) Aβ(1–40) (50 μM) peptides were dissolved and incubated in serial concentrations of bicine (78.130, 156.25, 312.50, 625.00, 1250.0, 2500.0, 5000.0, 10000, 20000 μM, presenting ln concentration) for 2 days at 37°C. (b) Aβ(1–40) (50 μM) peptides were dissolved and incubated in bicine (20 mM) or PBS (1X) at 3 time points: 2, 4 and 7 days. ThT fluorescence intensity was normalized to the Aβ(1–40) sample incubated in bicine for 7 days (100%). These experiments were performed in triplicate independently. Error bars indicate the standard deviation. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. ***P ≤ 0.001, ****P ≤ 0.0001. ThT: thioflavin T, A.U.: arbitrary unit, Conc.: concentration, d: day.
Fig 2.
Analysis of Aβ(1–40) peptides in bicine and PBS via (a) structural analysis by CD spectra and (b) TEM imaging. (a) Aβ(1–40) (25 μM) peptides were dissolved and incubated in bicine (20 mM) for 2 days or PBS (1X) for 0 and 2 days. (b) Aβ(1−40) (50 μM) incubated for 2 days in bicine (20 mM) and PBS. TEM imagery scale bar indicates 100 nm of size. These experiments were performed in triplicate independently. CD: circular dichroism, TEM: transmission electron microscopy, d: day.
Fig 3.
Assessing Aβ oligomeric aggregates via (a) electrophoresis and (b) MTT cell viability assay. Aβ(1–40) (50 μM) peptides were dissolved and incubated in bicine (20 mM) or PBS (1X) at 3 time points: 2, 4 and 7 days. The 0 day Aβ(1–40) sample was dissolved in PBS and immediately stored at −70°C before use. (a) Aβ aggregates were visualized by silver staining after SDS-PAGE with PICUP. Aβ samples in bicine and PBS were run on separate gels; original uncropped and unadjusted images of gels are provided in Supporting Information (S1 Fig). (b) Aβ aggregates were treated to HT-22 cells. Non-treated cells were used as a control. These experiments were performed in triplicate independently. Error bars indicate the standard deviation. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis, PICUP: photo-induced cross-linking of unmodified proteins, Ctrl: control, d: day.