Fig. 2. Cordycepin alleviated the ethanol-induced inhibition on osteogenesis of hBMSCs. (A-B) The ALP activity of hBMSCs was measured at 7 and 14 days. hBMSCs were cultured in osteogenic medium supplemented with ethanol and/or cordycepin as indicated. (Values are shown as the mean ± SE (N=3) *p<0.05) (C-D) The mRNA expression of BMP2 and OCN in hBMSCs after 24 hours incubation of ethanol and/or cordycepin. (Values are shown as the mean ± SE (N=3) *p<0.05) (E) hBMSCs were incubated for 72 hours. The RUNX2 level was decreased by ethanol and increased by cordycepin in hBMSCs. Proteins were immunoblotted with primary antibodies against RUNX2. β-actin served as a normalization control. (F) Proliferation of hBMSCs incubated for 1, 3, 5 and 7 days in medium supplemented with 50 mM ethanol and/or cordycepin as indicated. Cordycepin did not impair the survival and proliferation of hBMSCs. (G) hBMSCs were incubated with 50 mM ethanol and/or 10 μg/ml of cordycepin for 72 hours in osteogenic medium. Cells were immunostained with OCN and COL1. Cell skeleton was stained with phalloidine, and cell nuclei were stained with DAPI. (H) The ethanol-induced anti-osteogenic effect in hBMSCs was reversed by cordycepin. Cells were incubated for 21 days with osteogenic medium and stained with Alizarin red.