Figure 4
AMPK activation is required for osteogenic differentiation in hBMSCs independently of PKCĪ“. (A) Post-confluent hBMSCs were cultured in GM or ODM for the indicated times. Cell lysates were prepared and subjected to western blot analysis using the indicated antibodies. (B) Post-confluent hBMSCs were cultured in GM or ODM with 10 Ī¼M compound C, a specific inhibitor of AMPK, and then harvested after 14 days. The mRNA expression of ALP, RUNX2, and OCN was estimated by RT-PCR. (C) Post-confluent hBMSCs were cultured in GM or ODM with 10 Ī¼M compound C. After 7 days of osteogenic induction, ALP activity was determined by the ALP activity assay. (D) For quantitative determination, the absorbance at 405 nm of ALP reaction was measured and expressed as the fold change of treated cells over vehicle-treated cells in GM. (E) Post-confluent hBMSCs were cultured in GM or ODM with 10 Ī¼M compound C. Differentiated osteoblasts were stained with alizarin red S after 14 days of osteogenic induction. (F) For quantitative determination, the absorbance of alizarin red S was measured at 570 nm and expressed as the fold change of treated cells over vehicle-treated cells in GM. The representative images from three independent experiments are shown. Data shown are means Ā± S.E. (*P <0.05 versus vehicle-treated cells in ODM) of three independent experiments.