Fig. 5.

Astrocytic SIRT3 overexpression mitigates the neurotoxicity caused by LPS/IFN-γ-stimulated astrocytes. (A) Primary astrocytes were transfected with Flag- or human SIRT3-Flag expression constructs for 3 days, and then immunoblotting was performed. Successful transfection of Flag or SIRT3-Flag in cells was confirmed by immunoblotting using an anti-SIRT3 antibody. SIRT3 protein levels were highly increased in SIRT3-Flag-expressing cells compared with Flag-expressing cells. Tubulin was used as loading control. Data are presented as the mean±SD of 3 independent experiments. ***p<0.001 (unpaired Student’s t-test). (B, C) Flag- or SIRT3-Flag-expressing astrocytes were treated with LPS+IFN-γ (100 ng/ml+50 unit/ml) for 24 h and then cocultured with primary cortical neurons in transwell culture inserts. Cell viability was measured using a CMFDA staining (green; B) or CCK-8 assay (C) after a coculture period of 5 days. Data are presented as the mean±SD. **p<0.005 (unpaired Student’s t-test). Scale bars, 20 µm. (D) Flag- or SIRT3-Flag-expressing astrocytes were treated with LPS+IFN-γ (100 ng/ml+50 unit/ml) for 24 h, and then, Real Time-PCR was performed. Transcription levels of target genes are presented as the mean±SD. 18S rRNA was used to normalize changes in specific gene expression. *p<0.05; **p<0.005; ***p<0.001 (one-way ANOVA with Tukey’s post-hoc comparison test).