Figure 5

The effect of obif expression on osteoblastic markers. (A-D) Northern blot analyses of osteoblastic differentiation markers in MC3T3-E1 cells. Comparison between MC3T3-obif cells and MC3T3-control cells (A). The overexpression of obif was confirmed in MC3T3-obif cells. Since the coding sequence of obif is connected with an IRES sequence in the retrovirus construct, transcripts produced are longer than endogenous obif transcripts. The mRNA levels of osteoblastic differentiation markers, in particular bsp and ocn increased in MC3T3-obif cells. The comparison among control cells (MC3T3-sh147) and obif knocked down cells (MC3T3-sh292, -sh301) (B-D). At day 14, Osx transcripts decreased in obif knocked down cells whereas Runx2 transcripts were not influenced by obif level (B). At day 28, Runx2 was down-regulated in MC3T3-sh292 cells where we could detect only faint levels of obif mRNA. In MC3T3-shRunx2 cells, obif was down-regulated whereas Runx2 was not affected in MC3T3-sh292 cells at day 8 (C). At day 14 and 28, ocn were down-regulated in both MC3T3-sh292 and -sh301 cells (D). At day 42, ocn mRNA level in MC3T3-sh301 cells is almost the same as that in the control cells, but significantly lower in MC3T3-sh292 cells. (E) Semi-quantitative RT-PCR of osteoblastic differentiation markers in primary calvarial cells. The data presented are derived from three independent experiments.