Fig. 4. SIK1 modulates osteogenesis though CRTC1.

a SIK1-WT-FLAG, SIK1-T182A-FLAG, or the control plasmid (EV) were transfected to C2C12 cells. After stimulation with hBMP2 (500 ng/mL) for 15 min, cell lysates were prepared and subjected to Western blotting to assess CRTC1 phosphorylation. b Primary preosteoblasts were stimulated with hBMP2 in the presence of HG-9-91-01 for 20 min. Nuclear and cytoplasmic CRTC1 proteins were analyzed by Western blotting. Lamin B and α-tubulin were used as loading controls for nuclear and cytoplasmic extracts. c–h Primary preosteoblasts were co-transfected with SIK1 siRNA and CRTC1 siRNA. After culturing in osteogenic medium for three days, ALP activity assay c or real-time PCR analyses for ALP, OSX, OCN, and COL1A1 mRNA were performed d–f. ***p < 0.001; **p < 0.01; *p < 0.05. t-test