Figure 4

The role of obif in the osteoblastic differentiation of MC3T3-E1 cells. (A, B) Effect of obif expression on cell growth in the MC3T3-E1 cells. The proliferation rate decreased somewhat in MC3T3-obif compared with MC3T3-cont (A). Cells infected with retroviruses expressing shRNA against obif (MC3T3-sh292, MC3T3-sh301) showed almost the same level of cell growth with MC3T3-sh-cont (B). (C) Effect of obif expression on ALP activity in MC3T3-E1 cells cultured in the presence of ascorbic acid and β-glycerophosphate. (D) Effect of expression of shRNAs against obif in MC3T3-E1 cells. The reduction of obif expression caused a significant decrease of ALP activity at all time points. The decrease was proportionate to the strength of the shRNA's knockdown effects. (E-N) Mineral deposition visualized by Alizarin Red staining (E-L), and calcium contents measured by a colorimetric assay (M, N). The mineral deposition observed in MC3T3-obif cells and MC3T3-cont cells at days 18 and 24 (E-H). The mineral deposition observed in MC3T3-sh301 cells compared to MC3T3-sh-cont cells at days 30 and 46 (I-L). Scale bars = 2 mm. Calcium contents were significantly higher in MC3T3-obif cells than in MC3T3-cont cells both at day 18 and day 26 (M). Calcium deposition in MC3T3-sh292/301 and MC3T3-sh-cont cells at days 30 and 46 (N).