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. 2012 May 9;287(27):22654–22661. doi: 10.1074/jbc.M111.279091

FIGURE 3.

FIGURE 3.

Repeated TGF-β1 administration inhibits expression of osteoblast differentiation markers in MC3T3-E1 cells. A, MC3T3-E1 cells were cultured in α-MEM (1), OBM (2), OBM with a single administration of 0.1 ng/ml TGF-β1 (3), and OBM with repeated administration of 0.1 ng/ml TGF-β1 (4) for 72 h. ALP activity was visualized by ALP activity staining of cells. A single TGF-β1 administration induced ALP activity. However, repeated TGF-β1 administration suppressed ALP activity. B, confluent cells were treated for 72 h with OBM (1), OBM with a single administration of 0.1 ng/ml TGF-β1 (2), and OBM with repeated administration of 0.1 ng/ml TGF-β1 (3). Repeated TGF-β1 administration inhibited the expression of Alp (B), Igf-1 (C), and Oc (D). Expression of these genes was analyzed by qRT-PCR, and their mRNA levels were normalized to that of 18 S rRNA and measured in triplicate. Figures shown represent at least three independent experiments. Values represent mean ± S.E. (n = 4). Bonferroni correction for multiple comparisons was applied. *, p < 0.001; **, p < 0.01.