Figure 7.

Silencing of HSP60 reduces neurotoxicity of HEK293 cell lysates added to cocultures of neurons and microglia. HEK293 cells were transfected with siRNA A and B targeting HSP60. Untransfected cells (control) and cells transfected with nonsilencing siRNA (neg. siRNA) served as controls. A, Immunoblot of lysed cells probed with the SPA-807 antibody directed against HSP60. β-Actin served as a loading control. B, Densitometric analysis of the immunoblot signals after detection by chemiluminescence. C, Cocultures of neurons and microglia derived from wild-type mice were treated with cell lysates of transfected and untransfected HEK293 cells and were subsequently immunostained with NeuN to mark neurons, and with DAPI. Scale bar, 50 μm. D, Quantitation of NeuN-positive neurons as a percentage of all DAPI-positive cells in cocultures treated with HEK293 cell lysates. Six high-power fields per coverslip were analyzed. For each condition, experiments were performed in duplicates. The data shown are representative of four individual experiments. The results are presented as mean ± SD (*p < 0.001) for the comparison of neg. siRNA with siRNA HSPD1A and for the comparison of neg. siRNA with siRNA HSPD1B (one-way ANOVA and post hoc Bonferroni's test).