Fig. 1. SIK1 knockdown enhances osteogenesis in vitro and ex vivo.

a Primary preosteoblasts were treated with osteogenic medium containing β-GP and AA. The mRNA levels of SIK members were analyzed by real-time PCR. b–c siRNA-transfected cells were cultured in osteogenic medium. Cells were stained for ALP activity at day 3 (B). Matrix mineralization was evaluated by Alizarin Red staining at day 12, followed by stain extraction and spectrophotometry (C). d Primary preosteoblasts isolated from SIK1 KO or WT mice were cultured with osteogenic medium. Differentiated cells were subjected to Alizarin Red staining at day 14. e–g Calvariae from four-day-old mice were transfected with control siRNA or SIK1 siRNA and cultured with hBMP2 (200 ng/mL) for seven days ex vivo. Calvariae were stained for ALP (E) or analyzed for mRNA levels of SIK1 and ALP by real-time PCR (F). Calvarial sections were immunostained with anti-OCN. OCN-positive cells on bone surface around the sagittal suture were scored. Magnified images of the red boxes in upper panels are shown in bottom panels with arrows indicating OCN-positive cells (G). Scale bars, 200 μm in B and E and 50 μm in G. ***p < 0.001; **p < 0.01; *p < 0.05. t-test