Figure 6

Fulvestrant treatment reduces osteogenic induction by daidzein analogs through inhibition of early, middle, and/or late osteogenic transcription. Bone marrow-derived mesenchymal stem cells (BMSCs) or adipose-derived stromal/stem cells (ASCs) were cultured in charcoal dextran-stripped osteogenic differentiation medium (CDS-ODM) and concurrently treated with vehicle, 17β-estradiol (E2; 10 nM), daidzein (1 μM), or daidzein analog 2g or 2l (1 μM) and fulvestrant. After 14 days, (A) BMSCs and (C) ASCs were stained with alizarin red and eluted with cetylpyridinium chloride (CPC). Quantification of the stain was measured at 590 nm and normalized to vehicle-treated cells. (B) BMSCs and (D) ASCs were collected after 3, 7, or 14 days of treatment. RNA was isolated from the cells and reverse transcribed into cDNA. Analyses of osteogenic transcription factors were assessed by quantitative polymerase chain reaction. Expression values are normalized to undifferentiated cells set to 1.0. Bars, ± standard deviation. *P < 0.05 compared with fulvestrant-treated cells; **P < 0.01 compared with fulvestrant-treated cells;***P < 0.001 compared with fulvestrant-treated cells; ^###P < 0.001 compared with fulvestrant and E2-treated cells; ^ΨΨΨP < 0.001 compared with fulvestrant and daidzein-treated cells.