Figure 2. The effect of nephronectin 3′UTR on osteoblast differentiation.

(A) A nephronectin construct with a fragment of the nephronectin 3′UTR spanning the target sequence of miR-378 is shown (Npnt+3′). Cell lysate and conditioned medium from MC3T3-E1 cells stably transfected with Npnt+3′ were analyzed by western blot showing secretion of the product. (B) Cells transfected with the full-length nephronectin (Npnt-1 and Npnt-2), nephronectin containing the 3′UTR (Npnt+3′-1 and Npnt+3′-2), or with the control vector were grown for 10 days and stained for ALP expression. Multiple fields were examined for each construct, and stained cells were counted per field for quantitative analysis. (C) Typical micrographs of ALP expression are shown using two different phases (top and bottom panels) of the microscope.