Figure 4

HSP60 induces loss of Oli-neu cells in the presence of microglia expressing TLR4 in vitro . (A) Oli-neu cells alone or supplemented with neonatal microglia from C57BL/6 J mice were incubated with various doses of HSP60 or PBS (control), as indicated, for 3 d. Subsequently, cell cultures were stained with APC antibody to mark oligodendrocyte precursor cells. APC+ cells were quantified, and results were statistically expressed as relative viability of Oli-neu cells by setting the viability of control cells to 100%. (B) Oli-neu cells co-cultured with neonatal microglia derived from C57BL/6 J (WT) or TLR4^−/− mice were incubated with 10 μg/ml HSP60 or PBS (control), as indicated. 1 μg/ml LPS served as a positive control. After 3 d, co-cultures were fixed and stained with both APC antibody (red) and with IB4 (green) to mark oligodendrocyte precursor cells and microglia, respectively. Scale bar, 100 μm. (C) APC+ cells were quantified, and results were statistically expressed as relative viability of Oli-neu cells by setting the viability of control cells to 100%. (A, C) Results are presented as mean of 3 individual experiments with each condition performed in duplicate +/− SD. ***p < 0.001; ****p < 0.0001 (for the comparison of HSP60-treated groups with the control group in A; for the comparison of indicated groups in C; two-way ANOVA with Bonferroni-selected pairs).