Figure 7.

NP1-MNP stimulates osteoblast differentiation and expression of autophagy-related genes. (A) MC3T3-E1 cells were differentiated to osteoblasts in the presence of 30 μg/mL NP1-MNP or not (control), and samples were harvested for alkaline phosphatase enzyme activity at the indicated times (n = 3). (B) MC3T3-E1 cells were treated as in (A), and cells were stained for mineralization with Alizarin red S after 14 days. (C) Cells were treated and harvested as in (A) and analyzed for RNA expression of osteocalcin (OSC) by qRT-PCR. (D) Cells treated as in (A) were analyzed by Western blotting and probed with antibodies specific for the indicated proteins. Ubiquitin (Ub) was used as a loading control. (E) Cells were treated as in (A) and analyzed for RNA levels of autophagy genes using qRT-PCR. qRT-PCR results were normalized to Ubiquitin C and fold change calculated using the 2^(−ΔΔCt) method. *P < 0.05, **P < 0.01 (students-t test).