FIG. 4.

Depletion of Pbx1 by shRNA results in increased expression of osteoblast-related genes in mesenchymal cells. (A) to C) C3H10T1/2 cells were infected with ∼100 PFU/cell of shRNA-encoding recombinant lentivirus (nonsilencing [NS] or three different Pbx1-specific targets [T1, T2, and T3]). C3H10T1/2 cells were then treated with 100 ng/ml BMP2 or vehicle (PBS) for a period of 7 days. (A) Relative protein levels of Pbx1 in treated C3H10T1/2 cells were determined by Western blotting using an anti-Pbx1 antibody. (B) C3H10T1/2 cells treated with Pbx1-shRNA, demonstrating increased alkaline phosphatase activity. Scale bar, 500 μm. (C) Relative expression of osteoblast-related genes, monitored by RT-qPCR, was significantly increased in Pbx1-shRNA-infected C3H10T1/2 cells. (D and E) MC3T3-E1 cells were infected with shRNA-containing lentivirus (as described above) and treated with 280 μM ascorbic acid-5 mM β-glycerol phosphate for a period of 12 days to induce osteogenesis. (D) MC3T3-E1 cells treated with Pbx1-shRNA demonstrated increased alkaline phosphatase activity. (E) Relative expression of osteoblast-related genes in MC3T3-E1 cells was determined by RT-qPCR. Statistical significance was determined by one-way ANOVA followed by a Bonferroni posttest. Data are presented as the means from three experiments ± SEM.