Figure 1

Neurons in the presence of microglia lacking TLR4 are protected against HSP60-induced injury in vitro . (A) Lysates of co-cultures containing C57BL/6 J neurons and microglia incubated with 10 μg/ml HSP60, 10 μg/ml LPS, or PBS (control) for 3 d were analyzed by SDS-PAGE and immunoblotting with neuronal nuclei (NeuN) and synaptophysin (SyPh) antibodies. β-actin served as a loading control. Immunoblot signals were analyzed by densitometry and expressed as fold change to control. One representative experiment of three independent experiments is shown. (B) Neurons supplemented with C57BL/6 J microglia were incubated with various doses of HSP60 or PBS (control) for 3 d. Subsequently, cells were stained with NeuN Ab. NeuN+ cells were quantified. Results were statistically expressed as relative neuronal viability by setting the viability of control cells to 100%. (C- H) Neurons supplemented with C57BL/6 J (WT) or TLR4^−/− microglia were incubated with 10 μg/ml HSP60 or PBS (control) for 3 d, as indicated. 1 μg/ml LPS served as a positive control. (C) Co-cultures were stained with NeuN antibody (red) and with IB4 (green) to mark neurons and microglia, respectively. (D) NeuN+ cells were quantified. Results were statistically expressed as relative neuronal viability. (E) Co-cultures were stained with TUNEL assay (red) and (E, G) DAPI (blue). Scale bar, 100 μm. (F) Quantification of TUNEL+ cells/field in cultures displayed in E. (H) Quantification of DAPI+ nuclei displaying apoptotic hallmarks, as indicated by arrows in cultures shown in G. (B, D, F, H) Results are presented as mean of 3–4 individual experiments, each condition performed in duplicate +/− SD. **p < 0.01, ***p < 0.001, ****p < 0.0001 (comparison of HSP60-treated groups with control in B; comparison of indicated groups in D; comparison of HSP60- and LPS-treated groups with control in F and H; two-way ANOVA with Bonferroni-selected pairs).