Figure 2.

Promotion of osteoblast mineralization by metformin. (A–C) U2OS and MG63 cells were treated with 2.5, 5, and 10 μM of metformin or an equal volume of DMSO (0.1%) for 1 h (A) and (B and C) 20 days. ALP activity in cells was analyzed. Significant differences between metformin and DMSO control groups are indicated (*P < 0.05, a paired t-test). Experiments were performed in triplicate, and the error bars represent standard deviation. (B) Cell culture medium containing the proper amount of metformin was changed every other day. The cells were then fixed and stained by Alizarin Red S. (C) Alizarin red S-stained cells were extracted, and the optical density was measured with a microplate reader. Significant differences between metformin and DMSO control groups are indicated (*P < 0.05, a paired t-test). Experiments were performed in triplicate, and the error bars represent standard deviation. (D) For Western blotting analysis of protein markers of osteoblast differentiation, cells were treated with 5 μM of metformin or an equal volume of DMSO (0.1%) for 1 h. Cells were harvested, and the lysed proteins were resolved on SDS-PAGE and immunoblotted with specific antibodies.