Fig. 2. Overexpression of SIRT3 attenuates rotenone- or H2O2-induced neurotoxicity in differentiated SH-SY5Y cells. (A, B) Stable Flag- or human SIRT3-Flag-expressing cell lines were treated with RA (10 µM) for 8 days to generate differentiated SH-SY5Y cells. Flag- or SIRT3-Flag-expressing differentiated SH-SY5Y cells were treated with rotenone (50, 100, or 500 µM; A)/H2O2 (100, 500, or 1000 µM; B) for 24 h, and then CCK-8 analysis was performed. SIRT3-expressing differentiated SH-SY5Y cells exhibited significantly reduced rotenone- or H2O2-induced neuronal toxicity. Data are presented as the mean±SD of 3 independent experiments. *p<0.05; **p<0.005; ***p<0.001 (unpaired Student’s t-test). (C) CMFDA staining of rotenone (100 µM) or H2O2 (500 µM)-treated Flag- or SIRT3-Flag-expressing cells at 24 h. Then, CMFDA-positive neurons were counted under a fluorescence microscope. Data are presented as the mean±SD of 3. *p<0.05; **p<0.005 (unpaired Student’s t-test). Scale bars, 200 µm. (D) Protein levels of mitochondrial and cytosolic SIRT3 in Flag- or SIRT3-Flag-expressing differentiated SH-SY5Y cells. Both mitochondrial and cytosolic SIRT3 protein levels were significantly increased by SIRT3 expression. COX-IV (mitochondrial fraction) and actin (cytosolic fraction) were used for normalization. Data are presented as the mean±SD of 3 independent experiments. ***p<0.001 (unpaired Student’s t-test).
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