Figure 6.

Hsp27 reduces the generation of ROS by Neuro2a cells exposed to exogenous α-syn fibrils. Neuro-2a cells were incubated in PBS containing DHE (2 μm) before the addition of monomeric (M) or fibrillar (F) α-syn (20 μm) that had been pre-incubated in the absence or presence of Hsp27 (or the control proteins BSA and α-lac; each at 2 μm). The ratio of oxidized to reduced DHE before and after treatment was determined for 20 cells per treatment. A, example traces are shown for individual cells treated with monomeric α-syn, fibrillar α-syn, fibrillar α-syn preincubated with Hsp27 or Hsp27 alone. The fluorescence ratio before and after treatment was fitted by a linear regression function and the change in the rate of ROS production because of treatment was calculated as the difference in the gradient of the fitted lines. B, the -fold change in ROS production was then determined relative to a buffer only control (dashed line) and is reported as mean ± S.E. (n = 3 biological repeats, with each repeat an average of 20 cells). Data were analyzed via one-way ANOVA with a Bonferroni multiple comparison post hoc test, where *** denotes a significant (p < 0.001) difference compared with the fibril alone sample.