Fig. 3

Effects of Nampt-knocking down by shRNA on the differentiation of MC3T3-E1cells. Cells were seeded in 6-well plates and stably transduced with lentivirus with scramble RNA (scRNA) or Nampt shRNA for indicated days. a Representative images of MC3T3-E1 cells transduced with no RNA (control), scRNA, and Nampt shRNA. The cells were stained with ALP after 0, 1, and 3 days of differentiation. b Activity of ALP was performed after 4 days of differentiation using PNPP as a substrate. c Relative total NAD/NADH levels in differentiated MC3T3 cells transduced with lentivirus with scRNA controls, or Nampt shRNA or undifferentiated cells at 3 days using a commercial NADNADH assay kit. d Relative RT-PCR quantification of Runx2 gene expression in differentiated MC3T3-E1 cells transduced with lentivirus with scRNA controls, or Nampt shRNA or undifferentiated cells at 3 days. Runx2 expression was normalized to undifferentiated scRNA control cells. e Representative images of Western blotting showing the expression of Nampt and Runx2 in differentiated (at 3 days) MC3T3-E1 cells transduced with lentivirus with scRNA, or Nampt-shRNA. f Densitometry analysis of Nampt and Runx2 expression. Bar mean ± SD. **p < 0.01 vs. the scRNA control. All experiments were performed in triplicate