Figure 3

Daidzein analogs increase alkaline phosphatase activity and phosphate deposition in bone marrow-derived mesenchymal stem cells and adipose-derived stromal/stem cells. Bone marrow-derived mesenchymal stem cells (BMSCs; n = 6) and adipose-derived stromal/stem cells (ASCs; n = 6) were cultured in charcoal dextran-stripped osteogenic differentiation medium and simultaneously delivered vehicle, 17β-estradiol (E2; 10 nM), daidzein (1 μM), or daidzein analog 2g or 2l (1 μM). (A) After 3 days, cells were stained were fixed and stained with 5-bromo-4-chloro-3-indolyl phosphate. (B) To quantify alkaline phosphatase (ALP) activity, the number of positive pixels per image (for a total of five images/donor) was counted by Image J (Sigma, St. Louis, MO, USA). (C) After 14 days, cells were stained were fixed and stained with silver nitrate. (D) To quantify phosphate deposition after silver nitrate staining, the number of pixels per image (for a total of five images/donor) was counted by Image J. Images were acquired at 4× magnification. Scale bar represents 100 μm. Bars, ± standard deviation. ***P < 0.001 compared with vehicle-treated cells; ^#P < 0.05 compared with E2-treated cells; ^###P < 0.001 compared with E2-treated cells; ^ΨΨΨP < 0.001 compared with daidzein-treated cells.