Fig. 3.

Treatment of 21-day differentiated MC3T3-E1 cells with PF-562,271 results in alterations in expression of Wnt, BMP2 and Shh but has no effect on Runx2 or osterix mRNA levels. MC3T3-E1 cells were differentiated with ascorbic acid and β-glycerophosphate for 21 days as described in the Methods. Cells were then treated for 96 h with DMSO or various concentrations of PF-562,271, replacing the media containing drug every 2 days. At D7 post initiation of drug treatment RNA was collected, and qRT-PCR performed using murine specific primers to assess the expression of A) RUNX2, B) Osterix, C) Wnt3a, D) BMP2, or E) Shh. Graphs represent the mean relative expression normalized to β-actin levels and DMSO as a control condition for triplicate technical replicates in n = 3 independent biological replicates. * p < 0.05, ** p < 0.01. (F) Cellular protein lysates were also generated at D28 (following initiation of PF-562,271 treatment at D21 of MC3T3-E1 differentiation) and assessed for levels of Wnt3a or Shh by western blot analysis with β-actin used as a control for total protein loading.