ellipticity were proportional to the input concentrations of Ab1– 40. Prolonged incubation of the mixture with lower concentrations of Ab1–40 (0.002 mM and 0.01 mM) for 7 d resulted in a decrease in the absolute value of the ellipticity from 2 to 1. Such transition could represent a shift from random-coil to orderly structures [14]. Unlike other samples, when the Ab1–40 concentration reached 0.02 mM, the spectra exhibited a shape that is typical for b-sheet structures. After 7 d of incubation, the negative peak shifted from 206 nm (3 d) to 217 nm. This change denotes the formation of a large quantity of b-sheet structures [13,14]. Given that the difference CD spectra were obtained by subtracting the S100A9 signal, we believe the transition to b-sheet structure is induced by Ab1–40. Considering the lack of transition in the SDS PAGE measurements following the addition of Ab1–40, we believe that the S100A9/Ab1–40 interaction does not result in any change in S100A9 secondary structures. With prolonged incubation (9 d), minor distinctions were observed in the 200 to 230 nm wavelength range (Fig. 3). The two negative peaks that originate from a -helix structures showed a tendency to fuse. After incubation for 1 d, the negative peak that centered at 222 nm nearly disappeared and an enhancement in the amplitude of the peak at 208 nm was observed. After 3 d of incubation, there was a decline in the amplitude of the negative peak and a shift in the peak position to 209 nm. The two negative peaks became even less evident when the samples were maintained at 370C for 9 d. The distance between peaks decreased from 14 nm (208 nm to 222 nm) to 9 nm (210 nm to 219 nm). Taken together, the results from AFM, ThT and CD analyses suggest that S100A9 retains its secondary structures throughout development, and the conformational and structural changes were caused by amyloid formation from the Ab peptide. Effect of Ab1–40 on S100A9 cytotoxicity The effect of S100A9, Ab1–40 and their aggregates on the viability of SH-SY5Y neuroblastoma cells was assessed using the WST-1 assay. In viable cells, WST-1 undergoes reduction by Inflammation and Alzheimer’s Disease (AD) mitochondrial dehydrogenases (succinate-tetrazolium reductase system) to soluble formazan, which serves as an indicator of the number of metabolically active cells. The effect of Ab1–40 on S100A9-induced cytotoxicity was investigated by pre-mixing S100A9 with various concentrations of Ab1–40 and incubating the samples with SH-SY5Y neuroblastoma cells for 1 d to 3 d (Fig. 4). During the first day of incubation, at least 0.04 mM Ab1– 40 was required to inhibit the cell cytotoxicity of S100A9 (increasing cell survival to 90% from 70%). After 2 d of incubation, a 50% increase in cell viability (from 30% to 80%) was observed for Ab1–40 concentrations exceeding 0.2 mM (0.4 mM and 0.2 mM). Although the cell survival rate for all samples dropped greatly on the third day of incubation, the addition of excessive amounts of Ab1–40 (0.2 mM and 0.4 mM) increased the cell viability from 10% to 30%. We concluded that the cytotoxicity of S100A9 in SH-SY5Y cells is greatly suppressed by the presence of sufficient amounts of Ab1–40 peptide (more than 0.2 mM Ab1–40 for 0.02 mM S100A9). Characterization of the interaction between Ab1–40 and S100A9 To explore the interaction between S100A9 and Ab1–40 during incubation with cells, the mixtures and their corresponding controls (S100A9 and Ab1–40 only) were analyzed using sodium dodecyl sulfate (SDS) and native polyacrylamide gel electropho- resis (PAGE). No significant shifts in protein band positions were observed (Fig. 5). According to the bands generated by the control samples (containing only S100A8, A9, or culture media alone), the uppermost band corresponds to components of the media and the lower two bands represent the S100A9 monomer and homodimer, respectively. Because there was no change in the position of the S100A9 bands, it appears that S100A9 maintained its native structure during incubation with the cells. The structural stability of S100A9 was further verified by comparison with the samples exposed to harsh conditions (pH 3.0 and continuous shaking at 800 rpm). After 1 day of shaking at acidic pH, the band position of S100A9 shifted significantly, indicating the formation of more compact, faster migrating structures. A downward shift in band position corresponds to S100A9 amyloid formation, thereforem the absence of changes could indicate an absence of S100A9 amyloid formation during incubation with the cells. Figure 3. Difference CD spectra of S100A9 (0.2 mM) with the addition of various concentrations Ab1–40 peptide (0.002 mM to 0.1 mM) incubated at pH 7.4 and 370C for (a) 3 d; (b) 7 d. The difference CD spectra are obtained by subtracting the ellipticity of the mixture with the ones containing only S100A9 (0.2 mM). (c) Ellipticity of S100A9 (0.2 mM) and Ab1–40 (0.2 mM) mixture during 9 d of incubation at pH 7.4 and 370C. (d) Detailed features of panel c. doi:10.1371/journal.pone.0032953.g003 Figure 4. Measurements of NHSY5Y cell line viability by WST1 assay in the presence of 0.02 mM S100A9 and various S100A9/ Ab1–40 mixtures. The S100A9 concentration remained 0.02 mM. The concentration of Ab1–40 addition varied from 0.02 mM to 0.4 mM. In control experiments, the cells were incubated in DMEM cell culture buffer alone and the cell viability was equal to 100%. doi:10.1371/journal.pone.0032953.g004 PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e32953