Fig. 3: NAM-induced SIRT3 and FOXO3A activation mediates the upregulation of mitochondrial antioxidants. | Experimental & Molecular Medicine

Fig. 3: NAM-induced SIRT3 and FOXO3A activation mediates the upregulation of mitochondrial antioxidants.

From: Nicotinamide enhances osteoblast differentiation through activation of the mitochondrial antioxidant defense system

Fig. 3

a The mitochondrial fraction was isolated after 10 μM NAM treatment with osteogenic medium for 1 d. SIRT3 activity was determined with the mitochondrial fraction via a SIRT activity assay kit. b MC3T3-E1 cells were treated with 10 μM NAM in osteogenic medium for 4 and 7 d. The mRNA level of Sirt3 was determined by RT‒qPCR. c The transactivation activity of FOXO3A was measured in MC3T3-E1 cells transfected with FHRE-Luc after NAM treatment for 2 d. d Luciferase activity was determined after co-transfection of an FHRE-Luc plasmid and a control plasmid (pcDNA3.1) or with plasmids expressing FOXO3A and treatment with NAM for 2 d. e The acetylation level of FOXO3A was determined by immunoprecipitation (IP) with an anti-acetylated-lysine antibody followed by immunoblot analysis with an anti-FOXO3A antibody. f MC3T3-E1 cells were treated with the indicated concentrations of NAM for 4 d in osteogenic medium, and cell lysates were immunoblotted with p-FOXO3A (S253) and FOXO3A antibodies. g MC3T3-E1 cells were treated with NAM in osteogenic medium for 4 d, after which they were subjected to subcellular fractionation. The subcellular localization of FOXO3A was determined by immunoblot analysis. h MC3T3-E1 cells were transfected with siCtrl or siSirt3. The cells were cultured in osteogenic medium with or without 10 μM NAM for 4 d, and the cell lysates were immunoblotted with SIRT3, p-FOXO3A (S253) and FOXO3A antibodies. The data are expressed as the mean ± SD. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001. ns, not significant.

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