Fig. 6.

Lipocalin-2 (Lcn2) gene silencing prevents Lcn2 upregulation and release into the medium. Astrocyte cultures were transfected for 6 h with Lcn2 or scrambled siRNA and then treated with the Late AD oxysterol mixture 10 μM (Mix) for 12 h. After treatment, the medium was changed and astrocytes were incubated with fresh medium for 24 h. Transient Lcn2 gene knockdown was evaluated by (A) real-time RT-PCR and (B) Western blotting of both lysates and astrocyte conditioned media (ACM) samples. Data were normalized to the corresponding β-actin levels. Data are expressed as mean values ± SD from three different experiments as percentage change from control (one-way ANOVA). ****P < 0.0001, ***P < 0.001, and *P < 0.05 vs control; ####P < 0.0001 vs oxysterol treated. (C) Astrocyte morphology was examined by immunocytochemistry using a glial fibrillary acidic protein (GFAP) antibody (red) and nuclei were stained with Hoechst 33258 (blue). Representative images from three experiments are shown. Cells were imaged using an LSM800 confocal microscope (Zeiss, 40× objective; scale bar: 100 μm).