Fig. 6. Knock-down of Sirt3 in primary astrocytes enhances the neurotoxicity caused by LPS/IFN-γ-stimulated astrocytes. (A) Primary astrocytes were transfected with control siRNA (50 nM) or mouse Sirt3-specific siRNA (50 nM) for 3 days, and then immunoblotting was performed. Successful knockdown of control siRNA or Sirt3 siRNA in cells was confirmed by immunoblotting using an anti-SIRT3 antibody. SIRT3 protein levels were significantly decreased in Sirt3 siRNA-transfected 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) Control siRNA or Sirt3 siRNA-transfected astrocytes were treated with LPS+IFN-γ (100 ng/ml+50 unit/ml) for 24 h and then cocultured with primary cortical neurons using transwell culture inserts. Neuronal 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<0.05 (unpaired Student’s t-test). Scale bars, 20 µm. (D) Control siRNA or Sirt3 siRNA-transfected 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).
© Exp Neurobiol
