Figure 5.

r-Irisin promotes β-catenin expression to regulate osteoblast differentiation under simulated microgravity condition. (a) Real-time PCR and Western blot analysis of β-catenin and quantification of β-catenin protein level in primary osteoblasts after treatment with either 1 nM r-irisin or PBS for 48 h; n = 3. (b) Real-time PCR analysis of β-catenin expression in tibias of control and hind-limb unloading mice for 28 days in vivo (n = 4) or in primary osteoblasts after random position machine for 48 h in vitro (n = 3). (c) Real-time PCR analysis of β-catenin, Alp, and ColIα1 expressions in MC3T3-E1 cells after treatment with either β-catenin small interfering RNA (siRNA) (Si-β-cat) or siRNA negative control (NC) (Si-NC) for 48 h; n = 3. (d) Representative images of ALP staining and quantification of staining areas in MC3T3-E1 cells after treatment with either Si-β-cat or Si-NC for 48 h. Scale bar, 5 mm; n = 3. (e) Real-time PCR analysis of β-catenin in primary osteoblasts after treatment with either 1 nM r-irisin or PBS for 48 h under simulated microgravity; n = 3. (f) Western blot analysis and quantification of β-catenin protein level in primary osteoblasts after treatment with either 1 nM r-irisin or PBS for 48 h under simulated microgravity; n = 3. Gapdh was used as the internal control for mRNA. Ctr: control; SM: simulated microgravity; Si-β-cat: β-catenin siRNA; Si-NC: siRNA NC. All data are the mean ± SD. Statistical differences between the two groups were determined by the Student’s t-test; * p < 0.05, ** p < 0.01.