Fig. 5: NAM prevents ROS-induced mitochondrial and functional impairment in osteoblasts.

a ALP staining and ARS staining were performed after 10 μM NAM treatment with osteogenic medium in the presence or absence of 100 μM H₂O₂ for 5 and 12 days. b, c Quantification of each staining was performed by ImageJ. d H₂O₂-decreased DEGs restored by NAM were investigated by GO analysis in biological process on Day 10. The top 20 GO terms were selected and listed based on the adjusted P value and enrichment score. e, f Correlation analysis was performed on the genes in (d). The normalized correlation matrix was created to show the correlations between GO terms. The circular bar plots depict the ratios of genes associated with each GO term. g The mitochondrial OCR was evaluated after treatment with 10 μM NAM with or without 100 μM H₂O₂ for 7 d in osteogenic medium using an XF96 Extracellular Flux Analyzer. h–j The parameters calculated from the curved OCR plot are described in bar plots. k–l MC3T3-E1 cells were treated with 100 μM H₂O₂ alone or in combination with 10 μM NAM for 24 h. Immunostaining was performed using a γH2AX antibody (red) and DAPI (blue). To quantify the number of cells with γH2AX foci, the cells containing ≥10 foci were counted. m–p MC3T3-E1 cells were treated with 300 μM H₂O₂ in combination with or without 10 μM NAM. To detect H₂O₂-induced cell death, flow cytometry analysis after Annexin V and PI double staining was performed. q The percentages of each cell population are presented in a bar plot (n = 3). The data are expressed as the mean ± SD. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001. ns, not significant.